C5AR antagonists

ABSTRACT

Compounds are provided that are modulators of the C5a receptor. The compounds are substituted piperidines and are useful in pharmaceutical compositions, methods for the treatment of diseases and disorders involving the pathologic activation of C5a receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority benefit of U.S. application Ser. No.12/823,039, filed Jun. 24, 2010, and U.S. application Ser. No.13/072,616, filed Mar. 25, 2011, the contents of which are incorporatedherein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

The complement system plays a central role in the clearance of immunecomplexes and in immune responses to infectious agents, foreignantigens, virus infected cells and tumor cells. Inappropriate orexcessive activation of the complement system can lead to harmful, andeven potentially life-threatening consequences due to severeinflammation and resulting tissue destruction. These consequences areclinically manifested in various disorders including septic shock;myocardial, as well as, intestinal ischemia/reperfusion injury; graftrejection; organ failure; nephritis; pathological inflammation; andautoimmune diseases.

The complement system is composed of a group of proteins that arenormally present in the serum in an inactive state. Activation of thecomplement system encompasses mainly three distinct pathways, i.e., theclassical, the alternative, and the lectin pathway (V. M. Holers, InClinical Immunology: Principles and Practice, ed. R. R. Rich, MosbyPress; 1996, 363-391): 1) The classical pathway is acalcium/magnesium-dependent cascade, which is normally activated by theformation of antigen-antibody complexes. It can also be activated in anantibody-independent manner by the binding of C-reactive protein,complexed with ligand, and by many pathogens including gram-negativebacteria. 2) The alternative pathway is a magnesium-dependent cascadewhich is activated by deposition and activation of C3 on certainsusceptible surfaces (e.g. cell wall polysaccharides of yeast andbacteria, and certain biopolymer materials). 3) The lectin pathwayinvolves the initial binding of mannose-binding lectin and thesubsequent activation of C2 and C4, which are common to the classicalpathway (Matsushita, M. et al., J. Exp. Med. 176: 1497-1502 (1992);Suankratay, C. et al., J. Immunol. 160: 3006-3013 (1998)).

The activation of the complement pathway generates biologically activefragments of complement proteins, e.g. C3a, C4a and C5a anaphylatoxinsand C5b-9 membrane attack complexes (MAC), all which mediateinflammatory responses by affecting leukocyte chemotaxis; activatingmacrophages, neutrophils, platelets, mast cells and endothelial cells;and increasing vascular permeability, cytolysis and tissue injury.

Complement C5a is one of the most potent proinflammatory mediators ofthe complement system. (The anaphylactic C5a peptide is 100 times morepotent, on a molar basis, in eliciting inflammatory responses than C3a.)C5a is the activated form of C5 (190 kD, molecular weight). C5a ispresent in human serum at approximately 80 μg/ml (Kohler, P. F. et al.,J. Immunol. 99: 1211-1216 (1967)). It is composed of two polypeptidechains, α and β, with approximate molecular weights of 115 kD and 75 kD,respectively (Tack, B. F. et al., Biochemistry 18: 1490-1497 (1979)).Biosynthesized as a single-chain promolecule, C5 is enzymaticallycleaved into a two-chain structure during processing and secretion.After cleavage, the two chains are held together by at least onedisulphide bond as well as noncovalent interactions (Ooi, Y. M. et al.,J. Immunol. 124: 2494-2498 (1980)).

C5 is cleaved into the C5a and C5b fragments during activation of thecomplement pathways. The convertase enzymes responsible for C5activation are multi-subunit complexes of C4b, C2a, and C3b for theclassical pathway and of (C3b)₂, Bb, and P for the alternative pathway(Goldlust, M. B. et al., J. Immunol. 113: 998-1007 (1974); Schreiber, R.D. et al, Proc. Natl. Acad. Sci. 75: 3948-3952 (1978)). C5 is activatedby cleavage at position 74-75 (Arg-Leu) in the α-chain. Afteractivation, the 11.2 kD, 74 amino acid peptide C5a from theamino-terminus portion of the α-chain is released. Both C5a and C3a arepotent stimulators of neutrophils and monocytes (Schindler, R. et al.,Blood 76: 1631-1638 (1990); Haeffner-Cavaillon, N. et al., J. Immunol.138: 794-700 (1987); Cavaillon, J. M. et al., Eur. J. Immunol. 20:253-257 (1990)).

In addition to its anaphylatoxic properties, C5a induces chemotacticmigration of neutrophils (Ward, P. A. et al., J. Immunol. 102: 93-99(1969)), eosinophils (Kay, A. B. et al., Immunol. 24: 969-976 (1973)),basophils (Lett-Brown, M. A. et al., J. Immunol. 117: 246-252 1976)),and monocytes (Snyderman, R. et al., Proc. Soc. Exp. Biol. Med. 138:387-390 1971)). Both C5a and C5b-9 activate endothelial cells to expressadhesion molecules essential for sequestration of activated leukocytes,which mediate tissue inflammation and injury (Foreman, K. E. et al., J.Clin. Invest. 94: 1147-1155 (1994); Foreman, K. E. et al., Inflammation20:1-9 (1996); Rollins, S. A. et al., Transplantation 69: 1959-1967(2000)). C5a also mediates inflammatory reactions by causing smoothmuscle contraction, increasing vascular permeability, inducing basophiland mast cell degranulation and inducing release of lysosomal proteasesand oxidative free radicals (Gerard, C. et al., Ann. Rev. Immunol. 12:775-808 (1994)). Furthermore, C5a modulates the hepatic acute-phase geneexpression and augments the overall immune response by increasing theproduction of TNF-α, IL-1-β, IL-6, IL-8, prostaglandins and leukotrienes(Lambris, J. D. et al., In: The Human Complement System in Health andDisease, Volanakis, J. E. ed., Marcel Dekker, New York, pp. 83-118).

The anaphylactic and chemotactic effects of C5a are believed to bemediated through its interaction with the C5a receptor. The human C5areceptor (C5aR) is a 52 kD membrane bound G protein-coupled receptor,and is expressed on neutrophils, monocytes, basophils, eosinophils,hepatocytes, lung smooth muscle and endothelial cells, and renalglomerular tissues (Van-Epps, D. E. et al., J. Immunol. 132: 2862-2867(1984); Haviland, D. L. et al., J. Immunol. 154:1861-1869 (1995);Wetsel, R. A., Immunol. Leff. 44: 183-187 (1995); Buchner, R. R. et al.,J. Immunol. 155: 308-315 (1995); Chenoweth, D. E. et al., Proc. Natl.Acad. Sci. 75: 3943-3947 (1978); Zwirner, J. et al., Mol. Immunol.36:877-884 (1999)). The ligand-binding site of C5aR is complex andconsists of at least two physically separable binding domains. One bindsthe C5a amino terminus (amino acids 1-20) and disulfide-linked core(amino acids 21-61), while the second binds the C5a carboxy-terminal end(amino acids 62-74) (Wetsel, R. A., Curr. Opin. Immunol. 7: 48-53(1995)).

C5a plays important roles in inflammation and tissue injury. Incardiopulmonary bypass and hemodialysis, C5a is formed as a result ofactivation of the alternative complement pathway when human blood makescontact with the artificial surface of the heart-lung machine or kidneydialysis machine (Howard, R. J. et al., Arch. Surg. 123: 1496-1501(1988); Kirklin, J. K. et al., J. Cardiovasc. Surg. 86: 845-857 (1983);Craddock, P. R. et al., N. Engl. J. Med. 296: 769-774 (1977)). C5acauses increased capillary permeability and edema, bronchoconstriction,pulmonary vasoconstriction, leukocyte and platelet activation andinfiltration to tissues, in particular the lung (Czermak, B. J. et al.,J. Leukoc. Biol. 64: 40-48 (1998)). Administration of an anti-C5amonoclonal antibody was shown to reduce cardiopulmonary bypass andcardioplegia-induced coronary endothelial dysfunction (Tofukuji, M. etal., J. Thorac. Cardiovasc. Surg. 116: 1060-1068 (1998)).

C5a is also involved in acute respiratory distress syndrome (ARDS),Chronic Obstructive Pulmonary Disorder (COPD) and multiple organ failure(MOF) (Hack, C. E. et al., Am. J. Med. 1989:86:20-26; Hammerschmidt D Eet al. Lancet 1980; 1: 947-949; Heideman M. et al. J. Trauma 1984; 4:1038-1043; Marc, M M, et al., Am. J. Respir. Cell and Mol. Biol.,2004:31:216-219). C5a augments monocyte production of two importantpro-inflammatory cytokines, TNF-α and IL-1. C5a has also been shown toplay an important role in the development of tissue injury, andparticularly pulmonary injury, in animal models of septic shock(Smedegard G et al. Am. J. Pathol. 1989; 135: 489-497; Markus, S., etal., FASEB Journal (2001), 15: 568-570). In sepsis models using rats,pigs and non-human primates, anti-C5a antibodies administered to theanimals before treatment with endotoxin or E. coli resulted in decreasedtissue injury, as well as decreased production of IL-6 (Smedegard, G. etal., Am. J. Pathol. 135: 489-497 (1989); Hopken, U. et al., Eur. J.Immunol. 26: 1103-1109 (1996); Stevens, J. H. et al., J. Clin. Invest.77: 1812-1816 (1986)). More importantly, blockade or C5a with anti-C5apolyclonal antibodies has been shown to significantly improve survivalrates in a caecal ligation/puncture model of sepsis in rats (Czermak, B.J. et al., Nat. Med. 5: 788-792 (1999)). This model share many aspectsof the clinical manifestation of sepsis in humans. (Parker, S. J. etal., Br. J. Surg. 88: 22-30 (2001)). In the same sepsis model, anti-C5aantibodies were shown to inhibit apoptosis of thymocytes (Guo, R. F. etal., J. Clin. Invest. 106: 1271-1280 (2000)) and prevent MOF(Huber-Lang, M. et al., J. Immunol. 166: 1193-1199 (2001)). Anti-05aantibodies were also protective in a cobra venom factor model of lunginjury in rats, and in immune complex-induced lung injury (Mulligan, M.S. et al. J. Clin. Invest. 98: 503-512 (1996)). The importance of C5a inimmune complex-mediated lung injury was later confirmed in mice (Bozic,C. R. et al., Science 26: 1103-1109 (1996)).

C5a is found to be a major mediator in myocardial ischemia-reperfusioninjury. Complement depletion reduced myocardial infarct size in mice(Weisman, H. F. et al., Science 249: 146-151 (1990)), and treatment withanti-C5a antibodies reduced injury in a rat model of hindlimbischemia-reperfusion (Bless, N. M. et al., Am. J. Physiol. 276: L57-L63(1999)). Reperfusion injury during myocardial infarction was alsomarkedly reduced in pigs that were retreated with a monoclonal anti-C5aIgG (Amsterdam, E. A. et al., Am. J. Physiol. 268:H448-H457 (1995)). Arecombinant human C5aR antagonist reduces infarct size in a porcinemodel of surgical revascularization (Riley, R. D. et al., J. Thorac.Cardiovasc. Surg. 120: 350-358 (2000)).

C5a driven neutrophils also contribute to many bullous diseases (e.g.,bullous pemphigoid, pemphigus vulgaris and pemphigus foliaceus). Theseare chronic and recurring inflammatory disorders clinicallycharacterized by sterile blisters that appear in the sub-epidermal spaceof the skin and mucosa. While autoantibodies to keratinocytes located atthe cutaneous basement membranes are believed to underlie the detachmentof epidermal basal keratinocytes from the underlying basement membrane,blisters are also characterized by accumulation of neutrophils in boththe upper dermal layers and within the blister cavities. In experimentalmodels a reduction of neutrophils or absence of complement (total orC5-selective) can inhibit formation of sub-epidermal blisters, even inthe presence of high auto-antibody titers.

Complement levels are elevated in patients with rheumatoid arthritis(Jose, P. J. et al., Ann. Rheum. Dis. 49: 747-752 (1990); Grant, E. P.,et al., J. of Exp. Med., 196(11): 1461-1471, (2002)), lupus nephritis(Bao, L., et al., Eur. J. of Immunol., 35(8), 2496-2506, (2005)) andsystemic lupus erythematosus (SLE) (Porcel, J. M. et al., Clin. Immunol.Immunopathol. 74: 283-288 (1995)). C5a levels correlate with theseverity of the disease state. Collagen-induced arthritis in mice andrats resembles the rheumatoid arthritic disease in human. Mice deficientin the C5a receptor demonstrated a complete protection from arthritisinduced by injection of monoclonal anti-collagen Abs (Banda, N. K., etal., J. of Immunol., 2003, 171: 2109-2115). Therefore, inhibition of C5aand/or C5a receptor (C5aR) could be useful in treating these chronicdiseases.

The complement system is believed to be activated in patients withinflammatory bowel disease (IBD) and is thought to play a role in thedisease pathogenesis. Activated complement products were found at theluminal face of surface epithelial cells, as well as in the muscularismucosa and submucosal blood vessels in IBD patients (Woodruff, T. M., etal., J of Immunol., 2003, 171: 5514-5520).

C5aR expression is upregulated on reactive astrocytes, microglia, andendothelial cells in an inflamed human central nervous system (Gasque,P. et al., Am. J. Pathol. 150: 31-41 (1997)). C5a might be involved inneurodegenerative diseases, such as Alzheimer disease (Mukherjee, P. etal., J. Neuroimmunol. 105: 124-130 (2000); O'Barr, S. et al., J.Neuroimmunol. (2000) 105: 87-94; Farkas, I., et al. J. Immunol. (2003)170:5764-5771), Parkinson's disease, Pick disease and transmissiblespongiform encephalopathies. Activation of neuronal C5aR may induceapoptosis (Farkas I et al. J. Physiol. 1998; 507: 679-687). Therefore,inhibition of C5a and/or C5aR could also be useful in treatingneurodegenerative diseases.

There is some evidence that C5a production worsens inflammationassociated with atopic dermatitis (Neuber, K., et al., Immunology73:83-87, (1991)), and chronic urticaria (Kaplan, A. P., J. AllergyClin. Immunol. 114; 465-474, (2004).

Psoriasis is now known to be a T cell-mediated disease (Gottlieb, E. L.et al., Nat. Med. 1: 442-447 (1995)). However, neutrophils and mastcells may also be involved in the pathogenesis of the disease (Terui, T.et al., Exp. Dermatol. 9: 1-10; 2000); Werfel, T. et al., Arch.Dermatol. Res. 289: 83-86 (1997)). Neutrophil accumulation under thestratum corneum is observed in the highly inflamed areas of psoriaticplaques, and psoriatic lesion (scale) extracts contain highly elevatedlevels of C5a and exhibit potent chemotactic activity towardsneutrophils, an effect that can be inhibited by addition of a C5aantibody. T cells and neutrophils are chemo-attracted by C5a (Nataf, S.et al., J. Immunol. 162: 4018-4023 (1999); Tsuji, R. F. et al., J.Immunol. 165: 1588-1598 (2000); Cavaillon, J. M. et al., Eur. J.Immunol. 20: 253-257 (1990)). Additionally expression of C5aR has beendemonstrated in plasmacytoid dendritic cells (pDC) isolated from lesionsof cutaneous lupus erythematous and these cells were shown to displaychemotactic behavior towards C5a, suggesting that blockade of C5aR onpDC might be efficacious in reducing pDC infiltration into inflamed skinin both SLE and psoriasis. Therefore C5a could be an importanttherapeutic target for treatment of psoriasis.

Immunoglobulin G-containing immune complexes (IC) contribute to thepathophysiology in a number of autoimmune diseases, such as systemiclupus erthyematosus, rheumatoid arthritis, Sjogren's disease,Goodpasture's syndrome, and hypersensitivity pneumonitis (Madaio, M. P.,Semin. Nephroi. 19: 48-56 (1999); Korganow, A. S. et al., Immunity 10:451-459 (1999); Bolten, W. K., Kidney Int. 50: 1754-1760 (1996); Ando,M. et al., Curr. Opin. Pulm. Med. 3: 391-399 (1997)). These diseases arehighly heterogeneous and generally affect one or more of the followingorgans: skin, blood vessels, joints, kidneys, heart, lungs, nervoussystem and liver (including cirrhosis and liver fibrosis). The classicalanimal model for the inflammatory response in these IC diseases is theArthus reaction, which features the infiltration of polymorphonuclearcells, hemorrhage, and plasma exudation (Arthus, M., C. R. Soc. Biol.55: 817-824 (1903)). Recent studies show that C5aR deficient mice areprotected from tissue injury induced by IC (Kohl, J. et al., Mol.Immunol. 36: 893-903 (1999); Baumann, U. et al., J. Immunol. 164:1065-1070 (2000)). The results are consistent with the observation thata small peptidic anti-C5aR antagonist inhibits the inflammatory responsecaused by IC deposition (Strachan, A. J. et al., J. Immunol. 164:6560-6565 (2000)). Together with its receptor, C5a plays an importantrole in the pathogenesis of IC diseases. Inhibitors of C5a and C5aRcould be useful to treat these diseases.

DESCRIPTION OF RELATED ART

Only recently have non-peptide based C5a receptor antagonists beendescribed in the literature (e.g., Sumichika, H., et al., J. Biol. Chem.(2002), 277, 49403-49407). Non-peptide based C5a receptor antagonisthave been reported as being effective for treating endotoxic shock inrats (Stracham, A. J., et al., J. of Immunol. (2000), 164(12):6560-6565); and for treating IBD in a rat model (Woodruff, T. M., etal., J of Immunol., 2003, 171: 5514-5520). Non-peptide based C5areceptor modulators also have been described in the patent literature byNeurogen Corporation, (e.g., WO2004/043925, WO2004/018460,WO2005/007087, WO03/082826, WO03/08828, WO02/49993, WO03/084524); DompeS. P. A. (WO02/029187); and The University of Queenland (WO2004/100975).

There is considerable experimental evidence in the literature thatimplicates increased levels of C5a with a number of diseases anddisorders, in particular in autoimmune and inflammatory diseases anddisorders. Thus, there remains a need in the art for new small organicmolecule modulators, e.g., agonists, preferably antagonists, partialagonists, of the C5a receptor (C5aR) that are useful for inhibitingpathogenic events, e.g., chemotaxis, associated with increased levelsanaphylatoxin activity. The present invention fulfills this and otherneeds.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having theformula:

and pharmaceutically acceptable salts, hydrates and rotomers thereof;wherein

-   C¹ is selected from the group consisting of aryl and heteroaryl,    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S; and wherein said aryl and    heteroaryl groups are optionally substituted with from 1 to 3 R¹    substituents;-   C² is selected from the group consisting of aryl and heteroaryl,    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S; and wherein said aryl and    heteroaryl groups are optionally substituted with from 1 to 3 R²    substituents;-   C³ is selected from the group consisting of C₁₋₈ alkyl or    heteroalkyl, C₃₋₈ cycloalkyl, C₃₋₈ cycloalkyl-C₁₋₄ alkyl, aryl,    aryl-C₁₋₄ alkyl, heteroaryl, heteroaryl-C₁₋₄ alkyl, heterocycloalkyl    or heterocycloalkyl-C₁₋₄ alkyl, wherein the heterocycloalkyl group    or portion has from 1-3 heteroatoms selected from N, O and S, and    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S, and each C³ is optionally    substituted with from 1-3 R³ substituents;-   each R¹ is independently selected from the group consisting of    halogen, —CN, —R^(c), —CO₂R^(a), —CONR^(a)R^(b), —C(O)R^(a),    —OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)C(O)₂R^(c),    —NR^(a)—C(O)NR^(a)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)R^(b),    —OR^(a), and —S(O)₂NR^(a)R^(b); wherein each R^(a) and R^(b) is    independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, and is optionally substituted with one or two oxo;    each R^(c) is independently selected from the group consisting of    C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl,    heterocycloalkyl, aryl and heteroaryl, and wherein the aliphatic and    cyclic portions of R^(a), R^(b) and R^(c) are optionally further    substituted with from one to three halogen, hydroxy, methyl, amino,    alkylamino and dialkylamino groups; and optionally when two R¹    substituents are on adjacent atoms, are combined to form a fused    five or six-membered carbocyclic or heterocyclic ring;-   each R² is independently selected from the group consisting of    halogen, —CN, —NO₂, —R^(f), —CO₂R^(d), —CONR^(d)R^(e), —C(O)R^(d),    —OC(O)NR^(d)R^(e), —NR^(e)C(O)R^(d), —NR^(e)C(O)₂R^(f),    —NR^(d)C(O)NR^(d)R^(e), —NR^(d)C(O)NR^(d)R^(e), —NR^(d)R^(e),    —OR^(d), and —S(O)₂NR^(d)R^(e); wherein each R^(d) and R^(e) is    independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, and is optionally substituted with one or two oxo;    each R^(f) is independently selected from the group consisting of    C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl,    heterocycloalkyl, aryl and heteroaryl, and wherein the aliphatic and    cyclic portions of R^(d), R^(e) and R^(f) are optionally further    substituted with from one to three halogen, hydroxy, methyl, amino,    alkylamino and dialkylamino groups, and optionally when two R²    groups are on adjacent atoms, they are combined to form a five- or    six-membered ring;-   each R³ is independently selected from the group consisting of    halogen, —CN, —R^(i), CO₂R^(g), —CONR^(g)R^(h), —C(O)R^(g),    —C(O)R^(i), —OC(O)NR^(g)R^(h), —NR^(h)C(O)R^(g), —NR^(h)CO₂R^(i),    —NR^(g)C(O)NR^(g)R^(h), —NR^(g)R^(h), —OR^(g), —OR^(j),    —S(O)₂NR^(g)R^(h), —X⁴—R^(j), —NH—X⁴—R^(j), —O—X⁴—R^(j),    —X⁴—NR^(g)R^(h), —X⁴—NHR^(j), —X⁴—CONR^(g)R^(h),    —X⁴—NR^(h)C(O)R^(g), —X⁴—CO₂R^(g), —O—X⁴—CO₂R^(g), —NH—X⁴—CO₂R^(g),    —X⁴—NR^(h)CO₂R^(i), —O—X⁴—NR^(h)CO₂R^(i), —NHR^(j) and —NHCH₂R^(j),    wherein X⁴ is a C₁₋₄ alkylene; each R^(g) and R^(h) is independently    selected from hydrogen, C₁₋₈ alkyl or heteroalkyl, C₃₋₆ cycloalkyl    and C₁₋₈ haloalkyl, or when attached to the same nitrogen atom can    be combined with the nitrogen atom to form a four-, five- or    six-membered ring having from 0 to 2 additional heteroatoms as ring    members selected from N, O or S and is optionally substituted with    one or two oxo; each R^(i) is independently selected from the group    consisting of C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆    cycloalkyl, heterocycloalkyl, aryl and heteroaryl; and each R^(j) is    selected from the group consisting of C₃₋₆ cycloalkyl, imidazolyl,    pyrimidinyl, pyrrolinyl, piperidinyl, morpholinyl,    tetrahydrofuranyl, tetrahydropyranyl, and    S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and    cyclic portions of R^(g), R^(h), R^(i) and R^(j) are optionally    further substituted with from one to three halogen, methyl, CF₃,    hydroxy, C₁₋₄ alkoxy, C₁₋₄ alkoxy-C₁₋₄ alkyl, —C(O)O—C₁₋₈ alkyl,    amino, alkylamino and dialkylamino groups, and optionally when two    R³ groups are on adjacent atoms, they are combined to form a five-    or six-membered ring; and-   X is hydrogen or CH₃.

In addition to the compounds provided herein, the present inventionfurther provides pharmaceutical compositions containing one or more ofthese compounds, as well as methods for the use of these compounds intherapeutic methods, primarily to treat diseases associated with C5asignalling activity.

In yet another aspect, the present invention provides methods ofdiagnosing disease in an individual. In these methods, the compoundsprovided herein are administered in labeled form to a subject, followedby diagnostic imaging to determine the presence or absence of C5aR. In arelated aspect, a method of diagnosing disease is carried out bycontacting a tissue or blood sample with a labeled compound as providedherein and determining the presence, absence, or amount of C5aR in thesample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides structures and activity for representative compounds ofthe present invention. The compounds were prepared using methods asdescribed generally below, as well as methods provided in the Examples.

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviation and Definitions

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical, having the number of carbon atoms designated (i.e. C₁₋₈ meansone to eight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. The term “alkyl” in itsbroadest sense is also meant to include those unsaturated groups such asalkenyl and alkynyl groups. The term “alkenyl” refers to an unsaturatedalkyl group having one or more double bonds. Similarly, the term“alkynyl” refers to an unsaturated alkyl group having one or more triplebonds. Examples of such unsaturated alkyl groups include vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. The term “cycloalkyl” refers to hydrocarbonrings having the indicated number of ring atoms (e.g., C₃₋₆cycloalkyl)and being fully saturated or having no more than one double bond betweenring vertices. “Cycloalkyl” is also meant to refer to bicyclic andpolycyclic hydrocarbon rings such as, for example,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term“heterocycloalkyl” refers to a cycloalkyl group that contain from one tofive heteroatoms selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. The heterocycloalkyl may be a monocyclic, abicyclic or a polycylic ring system. Non limiting examples ofheterocycloalkyl groups include pyrrolidine, imidazolidine,pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin,dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine,thiomorpholine, thiomorpholine-5-oxide, thiomorpholine-S,S-oxide,piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone,tetrahydrofuran, tetrhydrothiophene, quinuclidine, and the like. Aheterocycloalkyl group can be attached to the remainder of the moleculethrough a ring carbon or a heteroatom.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingfour or fewer carbon atoms. Similarly, “alkenylene” and “alkynylene”refer to the unsaturated forms of “alkylene” having double or triplebonds, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH—CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the terms“heteroalkenyl” and “heteroalkynyl” by itself or in combination withanother term, means, unless otherwise stated, an alkenyl group oralkynyl group, respectively, that contains the stated number of carbonsand having from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. The heteroatom(s) O, N and S may be placed at any interiorposition of the heteroalkyl group.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent radical, saturated or unsaturated or polyunsaturated,derived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—, —O—CH₂—CH═CH—, —CH₂—CH═C(H)CH₂—O—CH₂— and—S—CH₂—C≡C—. For heteroalkylene groups, heteroatoms can also occupyeither or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy,alkyleneamino, alkylenediamino, and the like).

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively. Additionally, for dialkylaminogroups, the alkyl portions can be the same or different and can also becombined to form a 3-7 membered ring with the nitrogen atom to whicheach is attached. Accordingly, a group represented as —NR^(a)R^(b) ismeant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl andthe like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“C₁₋₄ haloalkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon group which can be a single ring ormultiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to five heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl groups include phenyl, naphthyl and biphenyl, whilenon-limiting examples of heteroaryl groups include pyridyl, pyridazinyl,pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl,benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl,isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl,thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines,benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl,isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl,thiazolyl, furyl, thienyl and the like. Substituents for each of theabove noted aryl and heteroaryl ring systems are selected from the groupof acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like).

The above terms (e.g., “alkyl,” “aryl” and “heteroaryl”), in someembodiments, will include both substituted and unsubstituted forms ofthe indicated radical. Preferred substituents for each type of radicalare provided below. For brevity, the terms aryl and heteroaryl willrefer to substituted or unsubstituted versions as provided below, whilethe term “alkyl” and related aliphatic radicals is meant to refer tounsubstituted version, unless indicated to be substituted.

Substituents for the alkyl radicals (including those groups oftenreferred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be avariety of groups selected from: -halogen, —OR′, —NR′R″, —SR′,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR', —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted C₁₋₈ alkyl, unsubstitutedheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted C₁₋₈ alkyl, C₁₋₈ alkoxy or C₁₋₈ thioalkoxy groups, orunsubstituted aryl-C₁₋₄ alkyl groups. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 3-, 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude 1-pyrrolidinyl and 4-morpholinyl. The term “acyl” as used byitself or as part of another group refers to an alkyl radical whereintwo substitutents on the carbon that is closest to the point ofattachment for the radical is replaced with the substitutent ═O (e.g.,—C(O)CH₃, —C(O)CH₂CH₂OR′ and the like).

Similarly, substituents for the aryl and heteroaryl groups are variedand are generally selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR',—R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′, —NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —N₃,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C₁₋₄ alkyl, andunsubstituted aryloxy-C₁₋₄ alkyl. Other suitable substituents includeeach of the above aryl substituents attached to a ring atom by analkylene tether of from 1-4 carbon atoms.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted C₁₋₆ alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “ionic liquid” refers to any liquid that contains mostly ions.Preferably, in the present invention, “ionic liquid” refers to the saltswhose melting point is relatively low (e.g., below 250° C.). Examples ofionic liquids include but are not limited to 1-butyl-3-methylimidazoliumtetrafluoroborate, 1-hexyl-3-methylimidazolium tetrafluoroborate,1-octyl-3-methylimidazolium tetrafluoroborate,1-nonyl-3-methylimidazolium tetrafluoroborate,1-decyl-3-methylimidazolium tetrafluoroborate,1-hexyl-3-methylimidazolium hexafluorophosphate and1-hexyl-3-methylimidazolium bromide, and the like.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. The compounds of the present invention may alsocontain unnatural proportions of atomic isotopes at one or more of theatoms that constitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.For example, the compounds may be prepared such that any number ofhydrogen atoms are replaced with a deuterium (²H) isotope.

II. Compounds

In one aspect, the present invention provides compounds having theformula I:

and pharmaceutically acceptable salts, hydrates and rotomers thereof;wherein

-   C¹ is selected from the group consisting of aryl and heteroaryl,    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S; and wherein said aryl and    heteroaryl groups are optionally substituted with from 1 to 3 R¹    substituents;-   C² is selected from the group consisting of aryl and heteroaryl,    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S; and wherein said aryl and    heteroaryl groups are optionally substituted with from 1 to 3 R²    substituents;-   C³ is selected from the group consisting of C₁₋₈ alkyl or    heteroalkyl, C₃₋₈ cycloalkyl, C₃₋₈ cycloalkyl-C₁₋₄ alkyl, aryl,    aryl-C₁₋₄ alkyl, heteroaryl, heteroaryl-C₁₋₄ alkyl, heterocycloalkyl    or heterocycloalkyl-C₁₋₄ alkyl, wherein the heterocycloalkyl group    or portion has from 1-3 heteroatoms selected from N, O and S, and    wherein the heteroaryl group has from 1-3 heteroatoms as ring    members selected from N, O and S, and each C³ is optionally    substituted with from 1-3 R³ substituents;-   each R¹ is independently selected from the group consisting of    halogen, —CN, —R^(c), —CO₂R^(a), —CONR^(a)R^(b), —C(O)R^(a),    —OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)C(O)₂R^(c),    —NR^(a)—C(O)NR^(a)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)R^(b),    —OR^(a), and —S(O)₂NR^(a)R^(b); wherein each R^(a) and R^(b) is    independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, and is optionally substituted with one or two oxo;    each R^(c) is independently selected from the group consisting of    C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl,    heterocycloalkyl, aryl and heteroaryl, and wherein the aliphatic and    cyclic portions of R^(a), R^(b) and R^(c) are optionally further    substituted with from one to three halogen, hydroxy, methyl, amino,    alkylamino and dialkylamino groups; and optionally when two R¹    substituents are on adjacent atoms, are combined to form a fused    five or six-membered carbocyclic or heterocyclic ring;-   each R² is independently selected from the group consisting of    halogen, —CN, —NO₂, —R^(f), —CO₂R^(d), —CONR^(d)R^(e), —C(O)R^(d),    —OC(O)NR^(d)R^(e), —NR^(e)C(O)R^(d), —NR^(e)C(O)₂R^(f),    —NR^(d)C(O)NR^(d)R^(e), —NR^(d)C(O)NR^(d)R^(e), —NR^(d)R^(e),    —OR^(d), and —S(O)₂NR^(d)R^(e); wherein each R^(d) and R^(e) is    independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, and is optionally substituted with one or two oxo;    each R^(f) is independently selected from the group consisting of    C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl,    heterocycloalkyl, aryl and heteroaryl, and wherein the aliphatic and    cyclic portions of R^(d), R^(e) and R^(f) are optionally further    substituted with from one to three halogen, hydroxy, methyl, amino,    alkylamino and dialkylamino groups, and optionally when two R²    groups are on adjacent atoms, they are combined to form a five- or    six-membered ring;-   each R³ is independently selected from the group consisting of    halogen, —CN, —R^(i), —CO₂R^(g), —CONR^(g)R^(h), —C(O)R^(g),    —C(O)R^(i), —OC(O)NR^(g)R^(h), —NR^(h)C(O)R^(g), —NR^(h)CO₂R^(i),    —NR^(g)C(O)NR^(g)R^(h), —NR^(g)R^(h), —OR^(g), —S(O)₂NR^(g)R^(h),    —NH—X⁴—R^(j), —X⁴—NR^(g)R^(h), —X⁴—NHR^(j), —X⁴—CONR^(g)R^(h),    —X⁴—NR^(h)C(O)R^(g), —X⁴—CO₂R^(g), —O—X⁴—CO₂R^(g), —NH—X⁴—CO₂R^(g),    —X⁴—NR^(h)CO₂W, —O—X⁴—NR^(h)CO₂W, —NHR^(j) and —NHCH₂R^(j), wherein    X⁴ is a C₁₋₄ alkylene; each R^(g) and R^(h) is independently    selected from hydrogen, C₁₋₈ alkyl or heteroalkyl, C₃₋₆ cycloalkyl    and C₁₋₈ haloalkyl, or when attached to the same nitrogen atom can    be combined with the nitrogen atom to form a four-, five- or    six-membered ring having from 0 to 2 additional heteroatoms as ring    members selected from N, O or S and is optionally substituted with    one or two oxo; each R^(i) is independently selected from the group    consisting of C₁₋₈ alkyl or heteroalkyl, C₁₋₈ haloalkyl, C₃₋₆    cycloalkyl, heterocycloalkyl, aryl and heteroaryl; and each R^(j) is    selected from the group consisting of C₃₋₆ cycloalkyl, imidazolyl,    pyrimidinyl, pyrrolinyl, piperidinyl, morpholinyl,    tetrahydrofuranyl, tetrahydropyranyl, and    S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and    cyclic portions of R^(g), R^(h), R^(i) and R^(j) are optionally    further substituted with from one to three halogen, methyl, CF₃,    hydroxy, C₁₋₄ alkoxy, C₁₋₄ alkoxy-C₁₋₄ alkyl, —C(O)O—C₁₋₈ alkyl,    amino, alkylamino and dialkylamino groups, and optionally when two    R³ groups are on adjacent atoms, they are combined to form a five-    or six-membered ring; and-   X is hydrogen or CH₃.

In formula I, the substituent C¹ is, in one embodiment, selected fromthe group consisting of phenyl, pyridyl, indolyl and thiazolyl, each ofwhich is optionally substituted with from 1 to 3 R¹ substituents.Preferably, each R¹ is independently selected from the group consistingof halogen, —CN, —R^(c), —NR^(a)R^(b) and —OR^(a), and wherein eachR^(a) and R^(b) is independently selected from hydrogen, C₁₋₈ alkyl, andC₁₋₈ haloalkyl, or when attached to the same nitrogen atom can becombined with the nitrogen atom to form a pyrrolidine ring; each R^(c)is independently selected from the group consisting of C₁₋₈ alkyl, C₁₋₈haloalkyl and C₃₋₆ cycloalkyl, and wherein the aliphatic and cyclicportions of R^(a), R^(b) and R^(e) are optionally further substitutedwith from one to three hydroxy, methyl, amino, alkylamino anddialkylamino groups; and optionally when two R¹ substituents are onadjacent atoms, are combined to form a fused five or six-memberedcarbocyclic ring. In selected embodiments of the invention, C¹ isselected from:

Returning to formula I, the substituents C² is, in one embodiment,selected from the group consisting of phenyl, naphthyl, pyridyl andindolyl, each of which is optionally substituted with from 1 to 3 R²substituents. Preferably, each R² is independently selected from thegroup consisting of halogen, —R^(f) and —OR^(d); wherein each R^(d) isindependently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl;each R^(f) is independently selected from the group consisting of C₁₋₈alkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl, heterocycloalkyl and heteroaryl,and wherein the aliphatic and cyclic portions of R^(d) and R^(f) areoptionally further substituted with from one to three halogen, hydroxy,methyl, amino, alkylamino and dialkylamino groups. In selectedembodiments of the invention, C² is selected from the group consistingof:

The substituent C³ is, in some embodiments, selected from the groupconsisting of C₃₋₆ alkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkylC₁₋₂alkyl,phenyl, pyridinyl, pyrazolyl, piperidinyl, pyrrolidinyl,piperidinylmethyl and pyrrolidinylmethyl, each of which is optionallysubstituted with from 1 to 3 R³ substituents. Preferably, each R³ isindependently selected from the group consisting of halogen, —R^(i),—CO₂R^(g), —CONR^(g)R^(h), —NR^(h)C(O)R^(g), —NR^(h)C(O)₂R^(i),—NR^(g)R^(h), —OR^(g), —X⁴—NR^(g)R^(h), —X⁴—CONR^(g)R^(h),—X⁴—NR^(h)C(O)R^(g), —NHR^(j) and —NHCH₂R^(j), wherein X⁴ is a C₁₋₃alkylene; each R^(g) and R^(h) is independently selected from hydrogen,C₁₋₈ alkyl, C₃₋₆ cycloalkyl and C₁₋₈ haloalkyl, or when attached to thesame nitrogen atom can be combined with the nitrogen atom to form a fiveor six-membered ring having from 0 to 1 additional heteroatoms as ringmembers selected from N, O or S and is optionally substituted with oneor two oxo; each R^(i) is independently selected from the groupconsisting of C₁₋₈ alkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl,heterocycloalkyl, aryl and heteroaryl; and each R^(j) is selected fromthe group consisting of C₃₋₆ cycloalkyl, pyrrolinyl, piperidinyl,morpholinyl, tetrahydrofuranyl, and tetrahydropyranyl, and wherein thealiphatic and cyclic portions of R^(g), R^(h), R^(i) and R^(j) areoptionally further substituted with from one to three halogen, methyl,CF₃, hydroxy, amino, alkylamino and dialkylamino groups. In selectedembodiments of the invention, C³ is selected from the group consistingof:

In other embodiments, C³ is selected from the group consisting of:

Returning to formula I, X is preferably H.

Subformulae of Formula I:

In one embodiment of the invention, compounds of formula I havesubformula Ia:

In a second embodiment of the invention, compounds of formula I havesubformula Ib:

In a third embodiment of the invention, compounds of formula I havesubformula Ic:

wherein X¹ is selected from the group consisting of N, CH and CR¹; thesubscript n is an integer of from 0 to 2; X² is selected from the groupconsisting of N, CH and CR²; and the subscript m is an integer of from 0to 2.

In a fourth embodiment of the invention, compounds of formula I havesubformula Id:

wherein X¹ is selected from the group consisting of N, CH and CR¹; thesubscript n is an integer of from 0 to 2; X² is selected from the groupconsisting of N, CH and CR²; and the subscript m is an integer of from 0to 2.

In a fifth embodiment of the invention, compounds of formula I havesubformula Ie:

wherein the subscript p is an integer of from 0 to 3; X¹ is selectedfrom the group consisting of N, CH and CR¹; the subscript n is aninteger of from 0 to 2; X² is selected from the group consisting of N,CH and CR²; and the subscript m is an integer of from 0 to 2.

In other selected embodiments, the compounds of the invention arerepresented by:

wherein the substituents R¹, R² and R³, and the subscript p all have themeanings provided with reference to formula I.

In still other selected embodiments, the compounds of the invention arerepresented by:

wherein the substituents R¹ and R³, and the subscript p all have themeanings provided with reference to formula I.

In a particularly preferred group of embodiments, the compounds of theinvention are represented by formula (Ie⁵) wherein R³ is a memberselected from the group consisting of —NR^(g)R^(h), —NHR^(j) and—NHCH₂R^(j), and each R^(g), R^(h) and R^(j) have the meanings providedwith reference to formula I.

In another particularly preferred group of embodiments, the compounds ofthe invention are represented by formula (Ie⁵) wherein R³ is a memberselected from the group consisting of —X⁴—NR^(g)R^(h), —X⁴—R^(j) and—X⁴—NR^(h)COR^(g), and each of X⁴, R^(g), R^(h) and R^(j) have themeanings provided with reference to formula I.

Compounds of the invention having formula I can exist in differentdiastereomeric forms, e.g., the substituents C¹ and C² in subformulae Iaand Ic can be cis to each other or trans to each other. As used herein,the terms cis or trans are used in their conventional sense in thechemical arts, i.e., referring to the position of the substituents toone another relative to a reference plane, e.g., a double bond, or aring system, such as a decalin-type ring system or a hydroquinolone ringsystem: in the cis isomer, the substituents are on the same side of thereference plane, in the trans isomer the substituents are on oppositesides. Additionally, different conformers are contemplated by thepresent invention, as well as distinct rotamers. Conformers areconformational isomers that can differ by rotations about one or more abonds. Rotamers are conformers that differ by rotation about only asingle a bond.

Preparation of Compounds

Those skilled in the art will recognize that there are a variety ofmethods available to synthesize molecules represented in the claims. Ingeneral, useful methods for synthesizing compounds represented in theclaims consist of four parts, which may be done in any order: Formationof the piperidine ring, installation of two amide bonds, andinstallation and/or modification of functional groups on C¹, C², and C³.

Several methods for the preparation of claimed compounds are illustratedbelow (eq. 1-6).

Equations 1-4 demonstrate some methods of forming the piperidine ring.Coupling at the 2-position of the pyridine ring can be accomplished viatransition metal mediated couplings as shown in eq. 1-2, or metalcatalyzed addition of an organometallic species such as the zincate ormagnesium salt (eq. 3). Subsequent to coupling at the 2-position,transition metal mediated hydrogenation of the pyridine ring yields thepiperidine ring system (eq. 1-3). Another method results in elaborationof a β-amino acid to a piperidine ring as described in eq. 4. Thoseskilled in the art will recognize that many synthetic methodologies canyield substituted piperidines, including C—C or C—N cyclization ofacyclic precursors via alkylation or ring-closing metathesis. Relativestereochemistry may be set by a variety of methods, including facialselectivity during the hydrogenation step. Absolute stereochemistry mayalso be set via a variety of methods, via the use of chiral ligands or achiral auxiliary, separation of chiral diasteroisomers, use of chiralstarting materials, or classical resolution. Compounds with 2,3-transstereochemistry may have the relative stereochemistry set during thepiperidine formation, or may be derived via epimerization of a 2,3-cispiperidine as illustrated in eq. 5.

Acylation of the piperidine ring is described in equation 6. In the caseof eq. 6, X may be choosen from an appropriate group such as OH, Cl andF, or from any group capable of activating a carbonyl group for additionof an amine (e.g., OSu, imidazole, etc.). Such couplings may be assistedby the use of inorganic or organic bases, activating agents such asHBTU, and also by catalysts, in particular by those catalysts known intha art which assist in the formation of amide bonds, such as DMAP,HOBT, etc. Suitable coupling partners include a carboxylic acid and apiperidine, an acyl fluoride and an amine and so forth. Those skilled inthe art will recognize that there are other possible combinations whichwill also result in the desired product.

A variety of methods described above have been used to prepare compoundsof the invention, some of which are described in the examples.

A family of specific compounds of particular interest having formula Iconsists of compounds, pharmaceutically acceptable salts, hydrates androtomers thereof, as set forth in FIG. 1. For the compounds of FIG. 1,any bond not illustrating at attached atom or group is meant to be amethyl group. For example,

is meant to illustrate

III. Pharmaceutical Compositions

In addition to the compounds provided above, compositions for modulatingC5a activity in humans and animals will typically contain apharmaceutical carrier or diluent.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The pharmaceutical compositions for the administration of the compoundsof this invention may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacyand drug delivery. All methods include the step of bringing the activeingredient into association with the carrier which constitutes one ormore accessory ingredients. In general, the pharmaceutical compositionsare prepared by uniformly and intimately bringing the active ingredientinto association with a liquid carrier or a finely divided solid carrieror both, and then, if necessary, shaping the product into the desiredformulation. In the pharmaceutical composition the active objectcompound is included in an amount sufficient to produce the desiredeffect upon the process or condition of diseases.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions and self emulsifications as described in U.S. PatentApplication 2002-0012680, hard or soft capsules, syrups, elixirs,solutions, buccal patch, oral gel, chewing gum, chewable tablets,effervescent powder and effervescent tablets. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents, antioxidants andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as cellulose, silicon dioxide, aluminumoxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example PVP, cellulose, PEG, starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. They may also be coated by the techniques described in theU.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, polyethyleneglycol (PEG) of various average sizes (e.g., PEG400, PEG4000) andcertain surfactants such as cremophor or solutol, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example peanut oil, liquid paraffin, or olive oil.Additionally, emulsions can be prepared with a non-water miscibleingredient such as oils and stabilized with surfactants such as mono- ordi-glycerides, PEG esters and the like.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. Oral solutions can be prepared in combination with, for example,cyclodextrin, PEG and surfactants.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols. Additionally, the compounds can be administeredvia ocular delivery by means of solutions or ointments. Still further,transdermal delivery of the subject compounds can be accomplished bymeans of iontophoretic patches and the like. For topical use, creams,ointments, jellies, solutions or suspensions, etc., containing thecompounds of the present invention are employed. As used herein, topicalapplication is also meant to include the use of mouth washes andgargles.

The compounds of this invention may also be coupled a carrier that is asuitable polymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of theinvention may be coupled to a carrier that is a class of biodegradablepolymers useful in achieving controlled release of a drug, for examplepolylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross linked or amphipathic block copolymers of hydrogels. Polymers andsemipermeable polymer matrices may be formed into shaped articles, suchas valves, stents, tubing, prostheses and the like. In one embodiment ofthe invention, the compound of the invention is coupled to a polymer orsemipermeable polymer matrix that is formed as a stent or stent-graftdevice.

IV. Methods of Treating Diseases and Disorders Modulated by C5a

The compounds of the invention may be used as agonists, (preferably)antagonists, partial agonists, inverse agonists, of C5a receptors in avariety of contexts, both in vitro and in vivo. In one embodiment, thecompounds of the invention are C5aR antagonist that can be used toinhibit the binding of C5a receptor ligand (e.g., C5a) to C5a receptorin vitro or in vivo. In general, such methods comprise the step ofcontacting a C5a receptor with a sufficient amount of one or more C5areceptor modulators as provided herein, in the presence of C5a receptorligand in aqueous solution and under conditions otherwise suitable forbinding of the ligand to C5a receptor. The C5a receptor may be presentin suspension (e.g., in an isolated membrane or cell preparation), in acultured or isolated cell, or in a tissue or organ.

Preferably, the amount of C5a receptor modulator contacted with thereceptor should be sufficient to inhibit C5a binding to C5a receptor invitro as measured, for example, using a radioligand binding assay,calcium mobilization assay, or chemotaxis assay as described herein.

In one embodiment of the invention, the C5a modulators of the inventionare used to modulate, preferably inhibit, the signal-transducingactivity of a C5a receptor, for example, by contacting one or morecompound(s) of the invention with a C5a receptor (either in vitro or invivo) under conditions suitable for binding of the modulator(s) to thereceptor. The receptor may be present in solution or suspension, in acultured or isolated cell preparation or within a patient. Anymodulation of the signal transducing activity may be assessed bydetecting an effect on calcium ion calcium mobilization or by detectingan effect on C5a receptor-mediated cellular chemotaxis. In general, aneffective amount of C5a modulator(s) is an amount sufficient to modulateC5a receptor signal transducing activity in vitro within a calciummobilization assay or C5a receptor-mediated cellular chemotaxis within amigration assay.

When compounds of the invention are used to inhibit C5areceptor-mediated cellular chemotaxis, preferably leukocyte (e.g.,neutrophil) chemotaxis, in an in vitro chemotaxis assay, such methodscomprise contacting white blood cells (particularly primate white bloodcells, especially human white blood cells) with one or more compounds ofthe invention. Preferably the concentration is sufficient to inhibitchemotaxis of white blood cells in an in vitro chemotaxis assay, so thatthe levels of chemotaxis observed in a control assay are significantlyhigher, as described above, than the levels observed in an assay towhich a compound of the invention has been added.

In another embodiment, the compounds of the present invention are usefulfor facilitating organ transplants. In this embodiment, the compoundscan be placed in a solution, with the organ prior to transplant.

In another embodiment, the compounds of the present invention furthercan be used for treating patients suffering from conditions that areresponsive to C5a receptor modulation. As used herein, the term“treating” or “treatment” encompasses both disease-modifying treatmentand symptomatic treatment, either of which may be prophylactic (i.e.,before the onset of symptoms, in order to prevent, delay or reduce theseverity of symptoms) or therapeutic (i.e., after the onset of symptoms,in order to reduce the severity and/or duration of symptoms). As usedherein, a condition is considered “responsive to C5a receptormodulation” if modulation of C5a receptor activity results in thereduction of inappropriate activity of a C5a receptor. As used herein,the term “patients” include primates (especially humans), domesticatedcompanion animals (such as dogs, cats, horses, and the like) andlivestock (such as cattle, pigs, sheep, and the like), with dosages asdescribed herein.

Conditions that can be treated by C5a modulation:

Autoimmune disorders—e.g., Rheumatoid arthritis, systemic lupuserythematosus, Guillain-Barre syndrome, pancreatitis, lupus nephritis,lupus glomerulonephritis, psoriasis, Crohn's disease, vasculitis,irritable bowel syndrome, dermatomyositis, multiple sclerosis, bronchialasthma, pemphigus, pemphigoid, scleroderma, myasthenia gravis,autoimmune hemolytic and thrombocytopenic states, Goodpasture's syndrome(and associated glomerulonephritis and pulmonary hemorrhage),immunovasculitis, tissue graft rejection, hyperacute rejection oftransplanted organs; and the like.

Inflammatory disorders and related conditions—e.g., Neutropenia, sepsis,septic shock, Alzheimer's disease, multiple sclerosis, stroke,inflammatory bowel disease (IBD), age-related macular degeneration (AMD,both wet and dry forms), inflammation associated with severe burns, lunginjury, and ischemia-reperfusion injury, osteoarthritis, as well asacute (adult) respiratory distress syndrome (ARDS), chronic pulmonaryobstructive disorder (COPD), systemic inflammatory response syndrome(SIRS), atopic dermatitis, psoriasis, chronic urticaria and multipleorgan dysfunction syndrome (MODS). Also included are pathologicsequellae associated with insulin-dependent diabetes mellitus (includingdiabetic retinopathy), lupus nephropathy, Heyman nephritis, membranousnephritis and other forms of glomerulonephritis, contact sensitivityresponses, and inflammation resulting from contact of blood withartificial surfaces that can cause complement activation, as occurs, forexample, during extracorporeal circulation of blood (e.g., duringhemodialysis or via a heart-lung machine, for example, in associationwith vascular surgery such as coronary artery bypass grafting or heartvalve replacement), or in association with contact with other artificialvessel or container surfaces (e.g., ventricular assist devices,artificial heart machines, transfusion tubing, blood storage bags,plasmapheresis, plateletpheresis, and the like). Also included arediseases related to ischemia/reperfusion injury, such as those resultingfrom transplants, including solid organ transplant, and syndromes suchas ischemic reperfusion injury, ischemic colitis and cardiac ischemia.Compounds of the instant invention may also be useful in the treatmentof age-related macular degeneration (Hageman et al, P.N.A.S. 102:7227-7232, 2005).

Cardiovascular and Cerebrovascular Disorders—e.g., myocardialinfarction, coronary thrombosis, vascular occlusion, post-surgicalvascular reocclusion, atherosclerosis, traumatic central nervous systeminjury, and ischemic heart disease. In one embodiment, an effectiveamount of a compound of the invention may be administered to a patientat risk for myocardial infarction or thrombosis (i.e., a patient who hasone or more recognized risk factor for myocardial infarction orthrombosis, such as, but not limited to, obesity, smoking, high bloodpressure, hypercholesterolemia, previous or genetic history ofmyocardial infarction or thrombosis) in order reduce the risk ofmyocardial infarction or thrombosis.

Diseases of Vasculitis—Vasculitic diseases are characterized byinflammation of the vessels. Infiltration of leukocytes leads todestruction of the vessel walls, and the complement pathway is believedto play a major role in initiating leukocyte migration as well as theresultant damage manifested at the site of inflammation (Vasculitis,Second Edition, Edited by Ball and Bridges, Oxford University Press, pp47-53, 2008). The compounds provided in the present invention can beused to treat leukoclastic vasculitis, Wegener's granulomatosis,microscopic polyangiitis, Churg-Strauss syndrome, Henoch-Schonleinpurpura, polyateritis nodosa, Rapidly Progressive Glomerulonephritis(RPGN), cryoglobulinaemia, giant cell arteritis (GCA), Behcet's diseaseand Takayasu's arteritis (TAK).

HIV infection and AIDS—C5a receptor modulators provided herein may beused to inhibit HIV infection, delay AIDS progression or decrease theseverity of symptoms or HIV infection and AIDS.

Neurodegenerative disorders and related diseases—Within further aspects,C5a antagonists provided herein may be used to treat Alzheimer'sdisease, multiple sclerosis, and cognitive function decline associatedwith cardiopulmonary bypass surgery and related procedures.

Cancers—The C5a antagonists provided herein are also useful for thetreatment of cancers and precancerous conditions in a subject. Specificcancers that can be treated include, but are not limited to, sarcomas,carcinomas, and mixed tumors. Exemplary conditions that may be treatedaccording to the present invention include fibrosarcomas, liposarcomas,chondrosarcomas, osteogenic sarcomas, angiosarcomas, lymphangiosarcomas,synoviomas, mesotheliomas, meningiomas, leukemias, lymphomas,leiomyosarcomas, rhabdomyosarcomas, squamous cell carcinomas, basal cellcarcinomas, adenocarcinomas, papillary carcinomas, cystadenocarcinomas,bronchogenic carcinomas, melanomas, renal cell carcinomas,hepatocellular carcinomas, transitional cell carcinomas,choriocarcinomas, seminomas, embryonal carcinomas, wilm's tumors,pleomorphic adenomas, liver cell papillomas, renal tubular adenomas,cystadenomas, papillomas, adenomas, leiomyomas, rhabdomyomas,hemangiomas, lymphangiomas, osteomas, chondromas, lipomas and fibromas.

In another embodiment, the compounds of the present invention are usefulin the treatment of cisplatin induced nephrotoxicity. In thisembodiment, compound treatment can alleviate the nephrotoxicity inducedby cisplatin chemotherapy of malignancies (Hao Pan et al, Am J PhysiolRenal Physiol, 296, F496-504, 2009).

In one embodiment of the invention, the compounds of the invention canbe used for the treatment of diseases selected from the group consistingof sepsis (and associated disorders), COPD, rheumatoid arthritis, lupusnephritis and multiple sclerosis.

Treatment methods provided herein include, in general, administration toa patient an effective amount of one or more compounds provided herein.Suitable patients include those patients suffering from or susceptibleto (i.e., prophylactic treatment) a disorder or disease identifiedherein. Typical patients for treatment as described herein includemammals, particularly primates, especially humans. Other suitablepatients include domesticated companion animals such as a dog, cat,horse, and the like, or a livestock animal such as cattle, pig, sheepand the like.

In general, treatment methods provided herein comprise administering toa patient an effective amount of a compound one or more compoundsprovided herein. In a preferred embodiment, the compound(s) of theinvention are preferably administered to a patient (e.g., a human)orally or topically. The effective amount may be an amount sufficient tomodulate C5a receptor activity and/or an amount sufficient to reduce oralleviate the symptoms presented by the patient. Preferably, the amountadministered is sufficient to yield a plasma concentration of thecompound (or its active metabolite, if the compound is a pro-drug) highenough to detectably inhibit white blood cell (e.g., neutrophil)chemotaxis in vitro. Treatment regimens may vary depending on thecompound used and the particular condition to be treated; for treatmentof most disorders, a frequency of administration of 4 times daily orless is preferred. In general, a dosage regimen of 2 times daily is morepreferred, with once a day dosing particularly preferred. It will beunderstood, however, that the specific dose level and treatment regimenfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination (i.e., other drugsbeing administered to the patient) and the severity of the particulardisease undergoing therapy, as well as the judgment of the prescribingmedical practitioner. In general, the use of the minimum dose sufficientto provide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using medical or veterinarycriteria suitable for the condition being treated or prevented.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment orpreventions of conditions involving pathogenic C5a activity (about 0.5mg to about 7 g per human patient per day). The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. Dosage unit forms will generallycontain between from about 1 mg to about 500 mg of an active ingredient.For compounds administered orally, transdermally, intravaneously, orsubcutaneously, it is preferred that sufficient amount of the compoundbe administered to achieve a serum concentration of 5 ng(nanograms)/mL-10 μg (micrograms)/mL serum, more preferably sufficientcompound to achieve a serum concentration of 20 ng-1 μg/ml serum shouldbe administered, most preferably sufficient compound to achieve a serumconcentration of 50 ng/ml-200 ng/ml serum should be administered. Fordirect injection into the synovium (for the treatment of arthritis)sufficient compounds should be administered to achieve a localconcentration of approximately 1 micromolar.

Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most disorders, adosage regimen of 4 times daily, three times daily, or less ispreferred, with a dosage regimen of once daily or 2 times daily beingparticularly preferred. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, and rate of excretion, drugcombination (i.e., other drugs being administered to the patient), theseverity of the particular disease undergoing therapy, and otherfactors, including the judgment of the prescribing medical practitioner.

In another aspect of the invention, the compounds of the invention canbe used in a variety of non-pharmaceutical in vitro and in vivoapplication. For example, the compounds of the invention may be labeledand used as probes for the detection and localization of C5a receptor(cell preparations or tissue sections samples). The compounds of theinvention may also be used as positive controls in assays for C5areceptor activity, i.e., as standards for determining the ability of acandidate agent to bind to C5a receptor, or as radiotracers for positronemission tomography (PET) imaging or for single photon emissioncomputerized tomography (SPECT). Such methods can be used tocharacterize C5a receptors in living subjects. For example, a C5areceptor modulator may be labeled using any of a variety of well knowntechniques (e.g., radiolabeled with a radionuclide such as tritium), andincubated with a sample for a suitable incubation time (e.g., determinedby first assaying a time course of binding). Following incubation,unbound compound is removed (e.g., by washing), and bound compounddetected using any method suitable for the label employed (e.g.,autoradiography or scintillation counting for radiolabeled compounds;spectroscopic methods may be used to detect luminescent groups andfluorescent groups). As a control, a matched sample containing labeledcompound and a greater (e.g., 10-fold greater) amount of unlabeledcompound may be processed in the same manner. A greater amount ofdetectable label remaining in the test sample than in the controlindicates the presence of C5a receptor in the sample. Detection assays,including receptor autoradiography (receptor mapping) of C5a receptor incultured cells or tissue samples may be performed as described by Kuharin sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998)John Wiley & Sons, New York.

The compounds provided herein may also be used within a variety of wellknown cell separation methods. For example, modulators may be linked tothe interior surface of a tissue culture plate or other support, for useas affinity ligands for immobilizing and thereby isolating, C5areceptors (e.g., isolating receptor-expressing cells) in vitro. In onepreferred application, a modulator linked to a fluorescent marker, suchas fluorescein, is contacted with the cells, which are then analyzed (orisolated) by fluorescence activated cell sorting (FACS).

In FIG. 1, structures and activity are provided for representativecompounds described herein. Activity is provided as follows for thebinding assay as described herein: +, 500 nM≦IC₅₀<2000 nM; ++, 50nM≦IC₅₀<500 nM; +++, 5 nM≦IC₅₀<50 nM; and ++++, IC₅₀<5 nM.

V. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Reagents and solvents used below can be obtained from commercial sourcessuch as Aldrich Chemical Co. (Milwaukee, Wis., USA). ¹H-NMR spectra wererecorded on a Varian Mercury 400 MHz NMR spectrometer. Significant peaksare provided relative to TMS and are tabulated in the order:multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet) and number of protons. Mass spectrometry results are reportedas the ratio of mass over charge, followed by the relative abundance ofeach ion (in parenthesis). In the examples, a single m/e value isreported for the M+H (or, as noted, M−H) ion containing the most commonatomic isotopes. Isotope patterns correspond to the expected formula inall cases. Electrospray ionization (ESI) mass spectrometry analysis wasconducted on a Hewlett-Packard MSD electrospray mass spectrometer usingthe HP1100 HPLC for sample delivery. Normally the analyte was dissolvedin methanol at 0.1 mg/mL and 1 microliter was infused with the deliverysolvent into the mass spectrometer, which scanned from 100 to 1500daltons. All compounds could be analyzed in the positive ESI mode, usingacetonitrile/water with 1% formic acid as the delivery solvent. Thecompounds provided below could also be analyzed in the negative ESImode, using 2 mM NH₄OAc in acetonitrile/water as delivery system.

The following abbreviations are used in the Examples and throughout thedescription of the invention:

EtOH: Ethanol

EtONa: Sodium ethoxide

THF: Tetrahydrofuran

TLC: Thin layer chromatography

MeOH: Methanol

Compounds within the scope of this invention can be synthesized asdescribed below, using a variety of reactions known to the skilledartisan. One skilled in the art will also recognize that alternativemethods may be employed to synthesize the target compounds of thisinvention, and that the approaches described within the body of thisdocument are not exhaustive, but do provide broadly applicable andpractical routes to compounds of interest.

Certain molecules claimed in this patent can exist in differentenantiomeric and diastereomeric forms and all such variants of thesecompounds are claimed.

The detailed description of the experimental procedures used tosynthesize key compounds in this text lead to molecules that aredescribed by the physical data identifying them as well as by thestructural depictions associated with them.

Those skilled in the art will also recognize that during standard workup procedures in organic chemistry, acids and bases are frequently used.Salts of the parent compounds are sometimes produced, if they possessthe necessary intrinsic acidity or basicity, during the experimentalprocedures described within this patent.

Example 1 Synthesis ofcis-1-(2-fluoro-6-methylbenzoyl)-2-phenylpiperidine-3-carboxylic acid(3-trifluoromethylphenyl)amide

a) Pd(PPh₃)₄ (3.0 g, 2.6 mmol) was added to a solution of2-chloro-3-carboxyethylpyridine (25 g, 134.7 mmol), phenylboronic acid(21.04 g, 172.6 mmol) and K₂CO₃ (55.1 g, 399 mmol) in 1,4-dioxane (200mL) and water (200 mL). The reaction mixture was heated at 100° C. for 2h. The solution was then cooled to room temperature and the dioxane wasremoved under reduced pressure. The resulting aqueous layer wasextracted with ethyl acetate, and the combined organic layers were dried(Na₂SO₄), filtered through celite, and concentrated under reducedpressure. The residue was purified by flash chromatography (SiO₂,10-100% EtOAc/hexanes) to get the 2-phenylpyridine derivative in 91%yield (27.98 g). LC-MS R_(t) (retention time): 2.45 min, MS: (ES) m/z228 (M+H⁺).

b) PtO₂ (800 mg, 3.52 mmol) was added to a solution of2-phenyl-nicotinic acid ethyl ester (20 g, 88 mmol, prepared in step aabove) in EtOH (60 mL) and concentrated HCl (15 mL). The reactionmixture was hydrogenated using a Parr shaker at 40-45 psi, for 1 h. Thereaction mixture was then filtered through celite, washed with EtOH, andthe filtrate was concentrated under reduced pressure. The residue wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃. Purification byflash chromatography (SiO₂, 0-20% MeOH/CH₂Cl₂) gave the desired productin 85% yield (17.4 g). LC-MS R_(t) (retention time): 1.73 min, MS: (ES)m/z 234 (M+H⁺).

c) Oxalyl chloride (3.2 mL, 30.75 mmol) was added to the solution of2-fluoro-6-methylbenzoic acid (3.79 g, 24.6 mmol) in CH₂Cl₂ (20 mL) in areaction flask at room temperature, followed by addition of a catalyticamount of DMF. The reaction was kept stirring for 2 h at roomtemperature. Solvent and excess oxalyl chloride were removed in vacuoand the residue was dried under high vacuum for 20 min. The resultingacid chloride was dissolved in dry CH₂Cl₂ (20 mL) and cooled to 0° C.followed by the addition of the piperidine made in step b (5.56 g, 20.5mmol) and Et₃N (8.6 mL, 61.5 mmol). The mixture was then allowed to warmto room temperature and stirred overnight. The reaction mixture wasdiluted with CH₂Cl₂ and water was added. The layers were separated andthe aqueous layer was extracted with CH₂Cl₂. The combined organic layerswere dried (MgSO₄) and concentrated under reduced pressure. The residuewas purified by flash chromatography (SiO₂, 10-35% EtOAc/hexanes) togive 7.47 g of the desired compound 99% yield). LC-MS R_(t) (retentiontime): 2.50 min and 2.58 min (two rotamers), MS: (ES) m/z 370 (M+H⁺).

d) Lithium aluminum hydride solution (2.0 M in THF, 8.2 mL, 16.4 mmol)was added to a solution of the ester from step c (2.98 g, 8.06 mmol) inTHF (100 ml) at 0° C. The resulting solution was kept stirring at 0° C.for 2 h at which time the reaction was completed. 15% Aqueous NaOH (625μL) was added drop wise to quench the reaction followed by H₂O (625 μL).To the cloudy colloidal mixture was added additional water (1.85 mL),and the mixture was kept stirring for 1 h at rt. The mixture was thenfiltered through a celite plug, and the filtrate was concentrated underreduced pressure. Purification by flash chromatography (SiO₂, 33-67%EtOAc/hexanes) gave 2.46 g of the desired product (93% yield). LC-MS:R_(t) (retention time):1.90 min and 2.09 min (two rotamers), MS: (ES)m/z 328 (M+H⁺).

e) A solution of the alcohol from step d (1.42 g, 4.33 mmol,) in aceticacid (65 ml) was added to a slurry of CrO₃ (2.61 g, 26.1 mmol) in H₂O(16 ml) at room temperature. The resulting mixture was kept stirring atroom temperature until the reaction was completed (90 min). The mixturewas filtered through a Celite plug and the filtrate was concentratedunder reduced pressure. Purification by flash chromatography (SiO₂,3-10% CH₂Cl₂:MeOH followed by 50-67% EtOAc/hexanes) gave 1.03 g of thedesired product (70% yield). LC-MS: R_(t) (retention time): 1.88 min and2.12 min (two rotamers), MS: (ES) m/z 342 (M+H⁺).

f) 3-Trifluoromethylaniline (16.2 mg, 0.1 mmol, 1.0 eq) was added to asolution of the acid prepared above (34.2 mg, 0.1 mmol) andtriethylamine (6 eq) in CH₂Cl₂ (1 mL). T3P (95.5 mg, 0.15 mmol) was thenslowly added and the solution was allowed to stir at room temperaturefor 1.5 h. The reaction mixture was diluted with CH₂Cl₂ (1 mL), washedwith 1 N aqueous HCl followed by saturated aqueous NaHCO₃. The organiclayer was separated, dried over anhydrous MgSO₄, and concentrated underreduced pressure Purification by flash chromatography (SiO₂, 5-40%EtOAc/hexanes) gave 35 mg (73% yield) of the product as a white solid.¹H NMR (400 MHz, CDCl₃) δ 1.22-2.45 (m, 8H), 2.93-3.32 (m, 3H),6.77-7.82 (m, 12H), 9.10 (s, 0.38H), 9.30 (s, 0.62; H). LC-MS: R_(t)(retention time)=2.88 min, MS: (ES) m/z 485 (M+H⁺).

Example 2 Synthesis ofN-(3-tert-butylphenyl)-1-(5-chloro-3-methylpicolinoyl)-2-phenylpiperidine-3-carboxamide

a) 2-Chloronicotinoyl chloride (1.05 eq) dissolved in anhydrousdichloromethane (0.5 M) was added to a solution of 3-tert-butylaniline(1 eq) and 2 M aq K₂CO₃ (2.2 eq) in anhydrous dichloromethane (0.5 M) at0° C. over a period of 30 min, and the reaction mixture was allowed tostir at room temperature for an additional 1.5 h. The layers wereseparated and the aqueous layer was extracted with dichloromethane. Thecombined organic layer was washed with brine, dried (MgSO₄), filteredand concentrated to give the desired amide as a foamy solid which wasused as such in the next step without further purification. MS: (ES) m/z289.1 (M+H⁺).

b) Pd(PPh₃)₄ (2-5 mol %) was added to a solution of the above pyridineamide (1 eq), phenylboronic acid (1.4 eq) and 2 M aq K₂CO₃ (2.4 eq) intoluene (0.7 M) and the reaction mixture was heated at 100° C. overnight (˜12 h). After cooling to room temperature, the reaction mixturewas filtered through celite and the celite plug was washed with EtOAc.The filtrate was diluted with water and extracted with EtOAc, dried(MgSO₄), filtered and concentrated and concentrated under reducedpressure. The residue was purified by automated flash chromatography(SiO₂, 10% to 100% gradient of EtOAc-hexanes) and dried in vacuo to givethe 2-phenyl-3-carboxyamidepyridine in 60-75% yield, MS: (ES) m/z 331.2(M+H⁺).

c) PtO₂ (10 mol %) was added to a solution of the 2-phenylpyridinederivative prepares above (1 eq) in EtOH and concentrated HCl (excess,4:1 ratio) and the reaction mixture was hydrogenated using a Parr shakerat 40-45 psi, for 1.5 h. It was filtered through celite, washed withEtOH, and the filtrate was concentrated. The residue was diluted withCH₂Cl₂ and washed with saturated aq NaHCO₃. The residue was thenpurified by automated flash chromatography (SiO₂, 1% to 30% gradient ofCH₂Cl₂-MeOH) and dried in vacuo to give the title compound in ˜85% yieldas a foamy solid. MS: (ES) m/z 337.2 (M+H⁺).

d) 5-Chloro-3-methylpicolinic acid (30 mg, 0.16 mmol) andN-(3-tert-butylphenyl)-2-phenylpiperidine-3-carboxamide (50 mg, 0.15mmol, prepared in step c above) were dissolved in anhydrous DMF (1 mL).N,N-Diisopropylethylamine (0.15 mL) was added at room temperaturefollowed by HCTU (67 mg, 0.16 mmol). After stirring 2 h at ambienttemperature, LC-MS and TLC indicated the completion of the reaction. Thereaction mixture was diluted with EtOAc (50 mL) and washed with 1 N HCl(20 mL), saturated NaHCO₃ (30 mL), and brine (30 mL) and the resultingsolution was concentrated under reduced pressure. The residue waspurified by preparative HPLC (20→95% gradient of MeCN—H₂O with 0.1% TFA)and the pure fractions were lyophilized to afford the title compound (50mg, 67% yield). HPLC retention time=2.88 minutes. ¹H NMR (400 MHz,CDCl₃) δ 8.42 (d, 1H, J=0.8 Hz), 7.97 (br, 1H), 7.59 (d, 1H, J=0.8 Hz),7.56 (d, 1H, J=7.6 Hz), 7.34 (m, 3H), 7.20 (m, 3H), 7.10 (d, 1H, J=7.6Hz), 6.61 (two sets of br, 1H), 3.12 (two sets of m, 2H), 2.94 (threesets of m, 1H), 2.36 (s, 3H), 2.20 (two sets of br, 2H), 1.74 (brcomplex, 2H), 1.29 (s, 9H). MS: (ES) m/z 490.2 (M+H⁺).

Example 3 Synthesis ofcis-1-(2-methylbenzoyl)-2-(3-fluorophenyl)piperidine-3-carboxylic acid(3-tert-butylphenyl)amide

a) To a mixture of N-(3-tert-butylphenyl)-2-chloronicotinamide (570.2mg, 2 mmol), 3-fluorophenylboronic acid (401.2 mg, 2.8 mmol), 3 mL oftoluene, and 1 mL of 2 N potassium carbonate in water was addedtetrakis(triphenylphosphine)palladium(0) (234.5 mg, 0.2 mmol). Themixture was then heated at 90° C. for 3 hour under nitrogen, before itwas cooled down to room temperature. The reaction mixture was thendiluted with 30 mL of water and 150 mL of EtOAc. The organic layer wasseparated, washed with brine, and dried (Na₂SO₄). The organic solventwas removed under reduced pressure and the residue was purified bysilica gel column (40% EtOAc in hexane) to giveN-(3-tert-butylphenyl)-2-(3-fluorophenyl)nicotinamide (691.4 mg, 99%).MS: (ES) m/z 394.5 (M+H⁺).

b) A mixture of N-(3-tert-butylphenyl)-2-(3-fluorophenyl)nicotinamide(501.2 mg, 1.4 mmol), platinum oxide (51.9 mg, 0.21 mmol), andconcentrated HCl (400 μL, 5.2 mmol) in 5 ml, of ethanol was stirredvigorously under hydrogen balloon overnight. The mixture was filtered,and the solids washed with 25 mL of methanol three times. The combinedsolution was dried under reduced pressure. To the residue was added 30mL of saturated sodium bicarbonate and 150 mL of EtOAc. The organiclayer was separated, and dried over sodium sulfate. Evaporation ofsolvent gave the crude 2-(3-fluorophenyl)piperidine-3-carboxylic acid(3-tert-butylphenyl)amide as a brown solid, which was taken on directlyto the next step. MS: (ES) m/z 355.7 (M+H⁺).

c) To a solution of 2-(3-fluorophenyl)piperidine-3-carboxylic acid(3-tert-butylphenyl)amide (prepared above, 177.3 mg, 0.5 mmol) in 2 mLof dichloromethane was added Et₃N (100 μL, excess), and 2-methylbenzoylchloride (92.3 mg, 0.6 mmol) at room temperature. The resulting solutionwas then stirred at this temperature until completion of the reaction(10 min.). The reaction mixture was then directly loaded onto a silicagel column, and was purified by using ISCO (30% EtOAc in hexane) to givethe final product2-(3-fluorophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylic acid(3-tert-butylphenyl)amide (151.2 mg, 64% yield). ¹H NMR (400 MHZ, CDCl₃,mixture of rotomers): δ 7.91 (s, 0.6H), 7.85 (s, 0.4H), 7.18-7.46 (m,9H), 7.11 (m, 1H), 6.95 (m, 1H), 6.67 (d, J=1.2 Hz, 1H), 3.36 (d, J=1.6Hz, 0.4H), 3.26 (d, J=1.6 Hz, 1H), 3.05 (m, 1H), 2.89 (t, J=1.2 Hz, 1H),2.45 (s, 1H), 2.02-2.40 (m, 4H), 1.70-1.84 (m, 3H), 1.44-1.64 (s, 1H),1.32 (s, 6H), 1.25 (s, 1H). MS: (ES) m/z 473.2 (MAI).

Example 4 Synthesis ofcis-1-(2-methylbenzoyl)-2-(2,2-dimethylpropyl)piperidine-3-carboxylicacid (3-tert-butylphenyl)amide

a) To a stirred solution of 2-bromonicotinic acid (1.01 g, 5 mmol)dissolved in anhydrous dichloromethane (8 mL) were added EDCI (1.34 g, 7mmol) and 3-tert-butylaniline (0.74 g, 5 mmol) at room temperature andthe reaction mixture was stirred for 12 hours. The mixture was thendiluted with dichloromethane, followed by saturated sodium bicarbonateand water wash. The dichloromethane layer was dried over anhydrousmagnesium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography to obtain2-bromo-N-(3-tert-butylphenyl)nicotinamide in 59% yield (950 mg). Rt:2.44 min (20-100-5 method). MS: (ES) m/z 333, 335 (MAI).

b) 2,2-Dimethylpropylmagnesium chloride (1 M-diethylether, 4.8 mL, 4.8mmol) was added to a suspension of copper cyanide (215 mg, 2.40 mmol) inTHF (6 mL) at −78° C. After stirring at the same temperature for 1 hour,2-bromo-N-(3-tert-butylphenyl)nicotinamide (200 mg, 0.601 mmol) wasadded all at once as a solid. The reaction mixture was gradually warmedto room temperature and the reaction was allowed to stir overnight.Saturated ammonium chloride solution and ethyl acetate was added, andthe reaction mixture was filtered through celite and rinsed with ethylacetate. The layers were separated and the product was extracted oncemore with ethyl acetate. The combined organic layers were washed withbrine and dried over anhydrous sodium sulfate. After removing thesolvent under reduced pressure, the crude material was purified usingsilica gel column chromatography using a gradient of 20%-50% ethylacetate in hexanes to yieldN-(3-tert-butylphenyl)-2-(2,2-dimethylpropyl)nicotinamide (168 mg, 0.517mmol, 86%). R_(f)=0.45 (toluene: ethyl acetate=2:1).

c) N-(3-tert-Butylphenyl)-2-(2,2-dimethylpropyl)nicotinamide (168 mg,0.517 mmol) was dissolved in ethanol (5 mL). Platinum oxide (11.6 mg,0.0511 mmol) was added followed by concentrated hydrochloric acid (250μL). The reaction mixture was hydrogenated using a Parr apparatus for1.5 hours at 45 psi. Analysis of the reaction mixture showed incompleteconversion, and the sequence was repeated one more time. Platinum oxidewas filtered off and the solvents were removed under reduced pressure.The crude material was neutralized using saturated sodium bicarbonatesolution and extracted with ethyl acetate. The organic layer was thenwashed with brine and dried over anhydrous magnesium sulfate. Removal ofsolvent under reduced pressure gave the crude2,3-cis-2-(2,2-dimethylpropyl)piperidine-3-carboxylicacid-(3-tert-butylphenyl)amide (153 mg) which was used in the next stepwithout further purification.

d) To a solution of2,3-cis-2-(2,2-dimethylpropyl)piperidine-3-carboxylicacid-(3-tert-butylphenyl)amide (84.8 mg, 0.257 mmol) in pyridine (415μL, 5.13 mmol) at room temperature was added 2-methylbenzoyl chloride(81.6 mg, 0.528 mmol) in chloroform (415 μL). A catalytic amount (notweighed) of dimethylaminopyridine was added to enhance the reaction andthe mixture was stirred for three days. Ethyl acetate and water was thenadded to the reaction mixture and the product was extracted with ethylacetate three times. The combined organic layers were dried overanhydrous magnesium sulfate. After removal of the solvent under reducedpressure the crude material was purified via silica gel chromatographyusing 10%-20% ethyl acetate in hexanes to give2,3-cis-2-(2,2-dimethylpropyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid-(3-tert-butylphenyl)amide (47.0 mg, 0.105 mmol, 41%). Rf=0.6(hexanes:ethyl acetate=2:1). Rt=3.16 min., 3.26 min. (compound exists asmixtures of several conformers. 20-100-5 method.). ¹H NMR (CDCl₃) δ 9.68(s, 1H), 9.43 (s, 1H), 8.33 (s, 1H), 8.28 (s, 1H)), 6.97-7.79 (m, 8H),5.48 (br, 1H), 5.39 (dd, J=4, 10 Hz, 1H), 5.33 (dd, J=6, 6 Hz, 1H), 3.38(ddd, J=4, 14, 14 Hz, 2H), 3.25 (dd, J=13, 13 Hz, 2H), 2.66 (dd, J=4,8.4 Hz, 1H), 2.63 (ddd, J=2.8, 2.8, 8 Hz, 1H), 2.50 (s, 9H), 2.40 (s,9H), 2.25 (s, 9H), 2.13 (s, 9H), 1.79-1.99 (m, 2H), 1.23-1.56 (m, 2H),1.32 (s, 9H), 1.07 (s, 9H), 1.06 (s, 9H), 0.97 (s, 9H), 0.95 (s, 9H).MS: (ES) m/z 449 (M+H).

Example 5 Synthesis ofcis-2-cyclopentyl-1-(2-methylbenzoyl)piperidine-3-carboxylic acid(3-tert-butylphenyl)amide

a) Cyclopentylzinc bromide (0.5 M, 6.5 mL, 3.26 mmol) was added to aroom temperature stirred solution of the 2-chloronicotinic acid methylester (400 mg, 2.33 mmol), CuI (19 mg, 0.1 mmol) and Pd(dppf)Cl₂ (42 mg,0.06 mmol) in anhydrous dimethylacetamide (1.7 mL) under nitrogen. Thereaction mixture was heated to 70° C. for 3.5 hours, cooled to roomtemperature, filtered through celite, and the cake was rinsed with ethylacetate. The filtrate was washed with water, brine, dried (MgSO₄),filtered and concentrated under reduced. The residue was purified byflash chromatography (SiO2, 10-100% EtOAc/hexanes) to get the desiredcompound in 83% yield (400 mg). LC-MS R_(t) (retention time): 1.87 min;MS: (ES) m/z 206 (M+H).

b) n-BuLi (1.47 mL, 3.68 mmol) was added to the 3-tert-butylaniline (580mg, 3.89 mmol) at −78° C. in dry THF (2 mL) under nitrogen and thesolution was allowed to stir at 0° C. for 10 minutes. The reactionmixture was re-cooled to −78° C. and 2-cyclopentyl-nicotinic acid methylester (400 mg, 1.94 mmol) dissolved in dry THF (2 mL) was added to it.The reaction mixture was allowed to attain 0° C. over a period of 2hours, quenched with saturated aqueous NH₄Cl, and extracted with ethylacetate. The combined organic layers were dried (MgSO₄), filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography (SiO2, 10-100% EtOAc/hexanes) to give the pure compoundin 91% yield (572 mg). LC-MS R_(t) (retention time): 2.61 min; MS: (ES)m/z 323 (M+H).

c) To a solution of the N-(3-tert-butylphenyl)-2-cyclopentylnicotinamide(570 mg, 1.77 mmol) in ethanol (10 mL) containing concentrated HCl (1mL) was added platinum oxide (40 mg, 0.17 mmol) and the solution washydrogenated using a Parr shaker at 40 psi for 1.5 hour. The reactionmixture was filtered through Celite, and the cake was rinsed withethanol. The filtrate was concentrated, and the residue was dried underhigh vacuum for 2 hours to get quantitative yield of the desiredpiperidine as a HCl salt. LC-MS R_(t) (retention time): 1.97 min; MS:(ES) m/z 329 (M+H⁺).

d) To a solution of the cis-2-cyclopentylpiperidine-3-carboxylic acid(3-tert-butyl-phenyl)amide prepared above (123 mg, 0.34 mmol) in dryCH₂Cl₂ (1 mL) containing Et₃N (142 μL, 1.02 mmol) was added2-methylbenzoyl chloride (53 mg, 0.34 mmol) and the mixture was stirredat room temperature for 2 hours. The reaction mixture was then dilutedwith ethyl acetate (20 mL), washed with 1 N aqueous HCl, water, andbrine. The organic layer was dried (MgSO₄), filtered and concentratedunder reduced pressure. The residue was purified by reverse phasepreparative HPLC (20-95% gradient of CH₃CN—H₂O) and dried (Lyophilizer)to give the title compound in 65% yield (109 mg). ¹H NMR (400 MHz,CDCl₃): δ 1.22-1.48 (m, 11H), 1.56-1.80 (m, 5H), 1.84-2.06 (m, 4H),2.10-2.23 (m, 1H), 2.30 (s, 1.6H), 2.39 (s, 1.4H), 2.41-2.50 (m, 1H),2.71-2.76 (m, 1H), 3.02-3.09 (m, 1H), 3.25-3.39 (m, 1H), 5.11 (bs, 1H),7.05-7.30 (m, 6H), 7.47-7.55 (m, 2H), 8.32 (bs, 1H). LC-MS R_(t)(retention time): 3.16 min; MS: (ES) m/z 447 (M+H)⁺. LC-MS method:Agilent Zorbax SB-C18, 2.1×50 mm, 5 g, 35° C., 1 mL/min flow rate, a 2.5min gradient of 20% to 100% B with a 1.0 min wash at 100% B; A=0.1%formic acid/5% acetonitrile/94.9 water, B=0.1% formic acid/5% water/94.9acetonitrile.

Example 6 Synthesis of(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid(3-chloro-4-methylphenyl)amide

b) cis-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylic acidethyl ester was synthesized similarly as illustrated in example 1.

c:1): cis-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylicacid ethyl ester (61 g, 174.8 mmol) and di-p-toluoyl-L-tartaric acid (62g, 174.8 mmol) was dissolved in EtOH (500 ml). The clear solution wasconcentrated and pumped to dry. The obtained white salt was thendissolved into 250 ml of ethyl acetate to form a clear solution. To thissolution was added 500 ml of TBME slowly. The obtained solution was leftat rt undisturbed for 3 days. At this time a lot of white crystals wereformed. They were then filtered and washed with 100 ml of TBME to obtaina white solid (60 g).

The above salt was re-dissolved in ethanol, concentrated and pumped todry. The obtained salt was dissolved into 500 ml of THF, followed byadding TBME (500 ml). The obtained clear solution was left at rtundisturbed for another 2.5 days. The obtained white crystals werefiltered to obtain 20.5 g (enrichment 64:1) of the salt.

c:2) To a 0° C. stirred suspension of the salt (16.7 g) in CH₂Cl₂ (150mL) was added saturated aqueous NaHCO₃ solution (100 mL) and thereaction mixture was allowed to stir at r.t over a period of 30 minutes.The layers were separated and the aqueous layer was extracted withCH₂Cl₂ (50 mL). The combined organic layer was washed with saturatedaqueous NaHCO₃ (2×100 mL), dried and concentrated to give(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylic acidethyl ester in 90% yield and ˜ in 97% ee.

d) To a 0° C. solution of the(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-piperidine-3-carboxylicacid ethyl ester prepared above (600 mg, 1.72 mmol) in dry CH₂Cl₂ (5 mL)containing Et₃N (480 μL, 3.44 mmol) was added 2-methylbenzoyl chloride(266 mg, 1.72 mmol) and the mixture was stirred at room temperature forover night. The reaction mixture was then diluted with CH₂Cl₂ (20 mL),washed with 1 N aqueous HCl, water, and brine. The organic layer wasdried (MgSO₄), filtered and concentrated under reduced pressure to give(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester in quantitative yield and the crude product was used assuch in the next step.

e) 4N HCl in 1,4-dioxane (5 mL, 20 mmol) was slowly added to a 0° C.solution of the above crude product(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester (840 mg, 1.72 mmol) in dry CH₂Cl₂ (4 mL). After theaddition of the HCl, the reaction mixture was allowed to attain r.t andstirred for 1 h. It was diluted with CH₂Cl₂ (30 mL), cooled to 0° C. andneutralized with saturated aqueous NaHCO₃ to get the(2R,3S)-2-(4-aminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester (612 mg) in 97% yield over two steps.

f) Na(OAC)₃BH (495 mg, 2.33 mmol) was added to a solution of the(2R,3S)-2-(4-aminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester (612 mg, 1.67 mmol), cyclopentanone (140 mg, 1.67 mmol)and acetic acid (100 mg, 1.67 mmol) in dry dichloroethane at r.t and thereaction mixture was heated to 50° C. for 4 h, cooled to r.t and stirredfor 48 h. It was then diluted with CH₂Cl₂ (30 mL), washed with saturatedaqueous NaHCO₃ solution, dried and concentrated in vacuo. The residuewas purified by ISCO flash column using ethyl acete and hexanes asmobile phase (40 g column, 0-40% gradient) to afford(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester (450 mg).

g) Me₃Al (290 μL, 0.57 mmol, 2M in toluene) was added to a solution ofthe 3-Chloro-4-methylphenylamine (65 mg, 0.46 mmol) in drydichloroethane (1 mL) at ambient temperature. Stirred for 20 minutes,then(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid ethyl ester (100 mg, 0.23 mmol) dissolved in dry dichloroethane (1mL) was added to it. The reaction mixture was then heated to 85° C. for3 h, cooled to r.t, diluted with CH₂Cl₂ (20 mL), washed with saturatedaqueous NaHCO₃ solution. The aqueous layer was extracted with CH₂Cl₂ (20mL) and the combined organic layer was dried (MgSO₄) and concentrated.The residue was purified by reverse phase preparative HPLC (20-95%gradient of CH₃CN—H₂O with 0.1% TFA as additive), the product containingfractions were pooled together and concentrated. The residue was dilutedwith CH₂Cl₂ (30 mL), washed with saturated aqueous NaHCO₃ solution. TheCH₂Cl₂ layer was dried (MgSO₄) and concentrated to get the pure(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylicacid (3-chloro-4-methylphenyl)amide in 50% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.4 (bs, 1H), 7.55 (s, 1H), 7.37-7.05 (m, 9H),6.55-6.52 (m, 2H), 3.77-3.70 (m, 1H), 3.30-3.16 (m, 1H), 3.04-2.91 (m,2H), 2.43-1.94 (m, 8H), 1.71-1.46 (m, 11H).

Example 7 Synthesis of ethyl(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylate

Step a) To a solution of(2R,3S)-2-(4-tert-butoxycarbonylaminophenyl)-piperidine-3-carboxylicacid ethyl ester (13.74 g, 39.36 mmol, prepared as in WO 2010/075257),2-fluoro-6-methylbenzoic acid (6.37 g, 41.33 mmol) and Et₃N (14.4 mL,102.3 mmol) in dry DMF (110 mL) at 0° C. was added HATU (15.71 g, 41.33mmol) and the mixture was then stirred at room temperature for 3 h. Thereaction mixture was then diluted with water, extracted with ethylacetate, and washed with brine. The combined organic layers were dried(MgSO₄), filtered, and concentrated under reduced pressure. The residuewas purified by flash chromatography (SiO₂, 10-55% ethyl acetate inhexanes) to give 19 g (100%) of the product ethyl(2R,3S)-2-[4-(tert-butoxycarbonylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylate.MS: (ES) m/z 485 (M+H⁺)

Step b) 4 N HCl in 1,4-dioxane (110 mL, 440 mmol) was slowly added to asolution of the above ethyl(2R,3S)-2-[4-(tert-butoxycarbonylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylate(19 g, 39.36 mmol) in dry CH₂Cl₂ (110 mL) at 0° C. After the addition ofHCl, the reaction mixture was allowed to attain rt and stirred for 3 h.The solution was then diluted with ethyl acetate (200 mL), cooled to 0°C., and neutralized with saturated aqueous NaHCO₃ to get ethyl(2R,3S)-2-(4-aminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylate(15 g, 100%). MS: (ES) m/z 385 (M+H⁺).

Step c) NaBH(OAc)₃ (14.12 g, 66.60 mmol) was added to a solution ofethyl(2R,3S)-2-(4-aminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylate(16 g, 41.65 mmol), and cyclopentanone (10.51 g, 125 mmol) in drydichloroethane (200 mL) at rt. The reaction mixture was heated to 45° C.for 3 h, cooled to rt, quenched with saturated aqueous NaHCO₃ solution,extracted with dichloromethane, dried and concentrated in vacuo. Theresidue was purified by flash chromatography (SiO₂, ethylacetate/hexanes 15-40%) to afford ethyl(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylateas white solid (17 g, 90%). MS: (ES) m/z 453 (M+H⁺).

Synthesis of(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)-N-[4-(hydroxymethyl)-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide

Step a) Ethyl(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methyl-benzoyl)piperidine-3-carboxylate(2 g, 4.4 mmol) in 1,4-dioxane (9 mL) was added to 3 N aqueous HCl (6mL) at room temperature and the reaction mixture was heated at 80° C.for 15 h. The solution was then cooled to 0° C. and neutralized withsaturated aqueous NaHCO₃ and extracted with ethyl acetate. The ethylacetate layer was concentrated under reduced pressure and the residuewas purified by flash chromatography (SiO₂, 20% EtOAc in CH₂Cl₂) to getthe desired acid (1.4 g, 74%). MS: (ES) m/z 425 (M+H⁺).

Step b) Methanesulfonyl chloride (378 mg, 3.3 mmol) was added to asolution of(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methyl-benzoyl)piperidine-3-carboxylicacid prepared above (1.4 g, 3.3 mmol) and Hunig's base (586 μL, 3.3mmol) in dry CH₂Cl₂ (25 mL) at 0° C. under a nitrogen atmosphere. Thesolution was stirred for an additional 15 minutes, then[4-amino-2-(trifluoromethyl)phenyl]methanol (631 mg, 3.3 mmol) andHunig's base (586 μL, 3.3 mmol) were added sequentially. The reactionmixture was allowed to attain room temperature over a period of 30minutes. When TLC and LCMS indicated completion of the reaction, excesssolvent was removed under reduced pressure. The residue was purified byflash chromatography (SiO2, 0-20% ethyl acetate in dichloromethane) toget the desired compound (1.2 g) in 61% yield. ¹H NMR (400 MHz, CDCl₃) δ9.50 (bs, 0.6H), 9.38 (bs, 0.4H), 7.75-7.70 (m, 1H), 7.50-7.33 (m, 3H),7.24-7.19 (m, 2H), 7.06-6.90 (m, 2H), 6.67-6.55 (m, 3H), 4.77-4.76 (m,2H), 3.78-3.66 (m, 1H), 3.32-3.00 (m, 3H), 2.44-1.45 (m, 17H). MS: (ES)m/z 598 (M+H⁺).

Example 8 Synthesis of4-[[(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methyl-benzoyl)piperidine-3-carbonyl]amino]-2-(trifluoromethyl)benzoicacid

The title compound was synthesized in similar fashion as the aboveexample: ¹H NMR (400 MHz, CD₃OD) δ 10.4 (br, 1H), 8.05-7.65 (m, 4H),7.36-6.95 (m, 5H), 6.50-6.47 (m, 1H), 3.92-3.94 (m, 1H), 3.50-3.21 (m,3H), 2.50-1.59 (m, 18H). MS: (ES) m/z 612 (M+H⁺).

Example 9 Synthesis ofcis-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)-N-[4-formyl-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide

To a solution ofcis-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methyl-benzoyl)-N-[4-(hydroxymethyl)-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide(130 mg, 0.22 mmol) in 1,2-dichloroethane (2 mL) was added MnO₂ (380 mg,4.4 mmol) at room temperature. The reaction mixture was stirred for 3 hand was then diluted with dichloromethane, filtered through a plug ofSiO2, and the filtrate was concentrated under reduced pressure. Theresidue was purified by HPLC to get the desired compound (as a mixtureof cis enantiomers) in 31% yield (40 mg). ¹H NMR (400 MHz, CDCl₃) δ 10.2(bs, 1H), 8.12-6.88 (m, 10H), 3.82-3.04 (m, 4H), 2.88-1.40 (m, 18H). MS:(ES) m/z 596 (M+H⁺).

Example 10(2R,3S)-2-(4-aminophenyl)-1-(2-fluoro-6-methylbenzoyl)-N-[4-methyl-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide

The title compound was synthesized in similar fashion as the aboveexamples: ¹HNMR (400 MHz, CDCl₃) δ 9.21 (br, 0.6H), 8.91 (br, 0.4H),7.67 (d, J=2.2 Hz, 0.4H), 7.60 (d, J=2.2 Hz, 0.6H), 7.51-7.47 (m, 1H),7.41 (d, J=8.4 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 7.24-7.03 (m, 2H),6.95-6.84 (m, 2H), 6.68 (d, J=5.5 Hz, 1H), 6.62-6.60 (m, 1H), 3.65 (br,2H), 3.28-2.96 (m, 3H), 2.44 (s, 1H), 2.41-2.38 (m, 3H), 2.11 (s, 3H),1.80-1.74 (m, 1H), 1.70 (s, 3H). MS: (ES) m/z 514 (M+H⁺)

Example 11

The following are representative compounds prepared and evaluated usingmethods similar to the examples herein. Characterization data isprovided for the compounds below. Biological evaluation is shown in FIG.1 for these compounds and others prepared as described herein.

(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (4-methyl-3-trifluoromethylphenyl)amide

¹H NMR (400 MHz, TFA-d) δ 7.91 (d, J=8.6 Hz, 1H), 7.84 (d, J=8.6 Hz,1H), 7.58-6.82 (m, 8H), 6.75 (t, J=8.6 Hz, 1H), 4.10-4.00 (m, 1H),3.60-3.47 (m, 1H), 3.45-3.41 (m, 1H), 3.33-3.25 (m, 1H), 2.44-2.22 (m,7H), 2.04-1.92 (m, 4H), 1.82-0.169 (m, 7H)

(2R,3S)-1-(2-Chlorobenzoyl)-2-(4-cyclopentylaminophenyl)piperidine-3-carboxylicacid (4-methyl-3-trifluoromethylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 9.41 (bs, 0.5H), 9.03 (bs, 0.5H), 7.55 (s,1H), 7.49-7.39 (m, 3H), 7.31-7.27 (m, 2H), 7.18-7.04 (m, 2H), 6.83-6.74(m, 3H), 3.76-3.64 (m, 1H), 3.22-2.90 (m, 5H), 2.39 (s, 3H), 2.32-2.20(m, 1H), 2.16-2.04 (m, 1H), 2.0-1.86 (m, 2H) 1.80-1.72 (m, 3H), 1.56(bs, 5H).

(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (3-chloro-4-methylphenyl)amide

¹H NMR (DMSO-d₆) δ 10.22 (s, 1H), 7.67 (dd, J=1.8 Hz, J=11.0 Hz, 1H),7.04-7.33 (m, 9H), 6.30 (dd, J=5.8 Hz, J=9.4 Hz, 1H), 5.52 (br, 1H),3.56-3.64 (m, 1H), 3.00-3.17 (m, 2H), 2.90-2.98 (m, 1H), 2.23 (2.24) (s,3H), 1.97 (2.33) (s, 3H), 1.32-2.22 (m, 12H)

(2R,3S)-1-(4-Chlorobenzoyl)-2-(4-Cyclopentylaminophenyl)piperidine-3-carboxylicacid (4-methyl-3-trifluoromethylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 8.79 (bs, 1H), 7.62 (s, 1H), 7.52-7.48 (m,1H), 7.37-7.30 (m, 5H), 7.13 (d, J=8.4 Hz, 1H), 6.52-6.50 (m, 3H),3.75-3.69 (m, 1H), 3.44 (bs, 1H), 3.09-2.97 (m, 2H), 2.39 (s, 3H),2.37-2.30 (m, 1H), 2.13-2.08 (m, 1H), 2.10-1.93 (m, 2H), 1.80-1.59 (m,7H), 1.48-1.42 (m, 2H)

(2R,3S)-2-(4-Cyclohexylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (3-^(t−)butylphenyl)amide

¹HNMR (400 MHz, CDCl₃): δ 8.24 (m, 1H), 7.40-6.85 (m, 8H), 6.65-6.40 (m,3H), 3.57 (s, 1H), 3.30-2.90 (m, 4H), 2.50-1.85 (m, 9H), 1.80-1.50 (m,5H), 1.40-1.00 (m, 13H)

(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (4-methyl-3-pyrrolidin-1-yl-phenyl)amide

¹H NMR (400 MHz, CDCl₃): δ 7.98 (m, 1H), 7.40-7.18 (m, 3H), 7.10-6.80(m, 4H), 6.64-6.40 (m, 3H), 3.80-3.50 (m, 2H), 3.30-2.90 (m, 6H),2.50-2.10 (m, 7H), 2.10-1.80 (m, 8H), 1.80-1.20 (m, 9H)

(2R,3S)-2-[4-(Cyclopentyloxy)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (3-chloro-4-methylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 8.68 (bs, 0.6H), 8.58 (bs, 0.4H), 7.59-7.40(m, 3H), 7.29-6.90 (m, 4H), 6.80 (m, 2H), 6.65 (m, 1H), 4.72 (m, 1H),3.30-2.92 (m, 3H), 2.44 (s, 1H), 2.42-2.30 (m, 1H), 2.30 (s, 1H), 2.29(s, 2H), 2.20 (s, 2H), 2.19-2.12 (m, 1H), 2.08-1.92 (m, 2H), 1.90-1.72(m, 7H) 1.60 (m, 2H).

(±)-(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (4-chloro-3-methylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 8.25 (bs, 0.4H), 8.16 (bs, 0.6H), 7.44-7.20(m, 6H), 7.06-6.84 (m, 2H), 6.59-6.50 (m, 2H), 3.75 (m, 1H), 3.66 (bs,1H), 3.26-2.92 (m, 3H), 2.43 (s, 1H), 2.42-2.30 (m, 1H), 2.30 (s, 1H),2.29 (s, 2H), 2.20 (s, 2H), 2.19-2.12 (m, 1H), 2.08-1.92 (m, 2H),1.80-1.58 (m, 7H) 1.45 (m, 2H).

(2R,3S)-2-(4-Cyclobutylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-3-carboxylicacid (3-^(t−)butylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 0.6H), 8.39 (s, 0.4H), 7.44-6.88 (m,10H), 6.25 (dd, J=12 Hz, J=6 Hz, 1H), 6.45 (t, J=8.4 Hz, 1H), 3.87 (m,1H), 3.26-2.95 (m, 3H), 2.46-2.05 (m, 8H), 1.86-1.61 (m, 5H), 1.34-1.11(m, 9H)

(2R,3S)-1-(2-fluoro-6-methylbenzoyl)-2-[4-(tetrahydropyran-4-ylamino)phenyl]piperidine-3-carboxylicacid (3-morpholin-4-yl-phenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 7.61 (s, 1H), 7.34-6.92 (m, 10H), 6.78-6.65(m, 1H), 6.62-6.53 (m, 1H), 3.98-3.85 (m, 4H), 3.83-3.70 (m, 1H),3.55-3.30 (m, 3H), 3.27-2.98 (M, 4H), 2.42-1.92 (m, 8H), 1.81-1.45 (m,7H)

(2R,3S)-1-(2-fluoro-6-methylbenzoyl)-2-[4-((R)-2-trffluoromethylpyrrolidin-1-ylmethyl)pheny]piperidine-3-carboxylicacid (3-^(t−)butylphenyl)amide

¹H NMR (400 MHz, CDCl₃) δ 8.01 (bs, 0.5H), 7.96 (bs, 0.5H), 7.55-7.37(m, 3H), 7.30-7.19 (m, 6H), 7.13-7.06 (m, 1H), 7.01-6.90 (m, 1H),6.85-6.64 (m, 1H), 4.15-4.11 (m, 1H), 3.58-3.54 (m, 1H), 3.30-3.20 (m,2H), 3.17-2.80 (m, 2H), 2.45-2.17 (m, 4H), 2.00-1.94 (m, 2H), 1.86-1.60(m, 8H), 1.31-1.26 (m, 7H)

Example 12 Materials and Methods

A. Cells

1. C5a receptor expressing cells

a) U937 Cells

U937 cells are a monocytic cell line which express C5aR, and areavailable from American Tissue Cell Collection (Virginia). These cellswere cultured as a suspension in RPMI-1640 medium supplemented with 2 mML-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1mM sodium pyruvate, and 10% FBS. Cells were grown under 5% CO₂/95% air,100% humidity at 37° C. and subcultured twice weekly at 1:6 (cells werecultured at a density range of 1×10⁵ to 2×10⁶ cells/mL) and harvested at1×10⁶ cells/mL. Prior to assay, cells are treated overnight with 0.5 mMof cyclic AMP (Sigma, Ohio) and washed once prior to use. cAMP treatedU937 cells can be used in C5aR ligand binding and functional assays.

b) Isolated Human Neutrophils

Optionally, human or murine neutrophils can be used to assay forcompound activity. Neutrophils may be isolated from fresh human bloodusing density separation and centrifugation. Briefly, whole blood isincubated with equal parts 3% dextran and allowed to separate for 45minutes. After separation, the top layer is layered on top of 15 mls ofFicoll (15 mls of Ficoll for every 30 mls of blood suspension) andcentrifuged for 30 minutes at 400×g with no brake. The pellet at thebottom of the tube is then isolated and resuspended into PharmLyse RBCLysis Buffer (BD Biosciences, San Jose, Calif.) after which the sampleis again centrifuged for 10 minutes at 400×g with brake. The remainingcell pellet is resuspended as appropriate and consists of isolatedneutrophils.

B. Assays

1. Inhibition of ¹²⁵I-C5a Binding to C5aR

cAMP treated U937 cells expressing C5aR were centrifuged and resuspendedin assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1 mM CaCl₂, 5 mMMgCl₂, and with 0.1% bovine serum albumin) to a concentration of 3×10⁶cells/mL. Binding assays were set up as follows. 0.1 mL of cells wasadded to the assay plates containing 5 μL of the compound, giving afinal concentration of ˜2-10 μM each compound for screening (or part ofa dose response for compound IC₅₀ determinations). Then 0.1 mL of ¹²⁵Ilabeled C5a (obtained from Perkin Elmer Life Sciences, Boston, Mass.)diluted in assay buffer to a final concentration of ˜50 μM, yielding30,000 cpm per well, was added, the plates sealed and incubated forapproximately 3 hours at 4° C. on a shaker platform. Reactions wereaspirated onto GF/B glass filters pre-soaked in 0.3% polyethyleneimine(PEI) solution, on a vacuum cell harvester (Packard Instruments;Meriden, Conn.). Scintillation fluid (40 μl; Microscint 20, PackardInstruments) was added to each well, the plates were sealed andradioactivity measured in a Topcount scintillation counter (PackardInstruments). Control wells containing either diluent only (for totalcounts) or excess C5a (1 μg/mL, for non-specific binding) were used tocalculate the percent of total inhibition for compound. The computerprogram Prism from GraphPad, Inc. (San Diego, Calif.) was used tocalculate IC₅₀ values. IC₅₀ values are those concentrations required toreduce the binding of radiolabeled C5a to the receptor by 50%. (Forfurther descriptions of ligand binding and other functional assays, seeDairaghi, et al., J. Biol. Chem. 274:21569-21574 (1999), Penfold, etal., Proc. Natl. Acad. Sci. USA. 96:9839-9844 (1999), and Dairaghi, etal., J. Biol. Chem. 272:28206-28209 (1997)).

2. Calcium Mobilization

Optionally, compounds may be further assayed for their ability toinhibit calcium flux in cells. To detect the release of intracellularstores of calcium, cells (e.g., cAMP stimulated U937 or neutrophils) areincubated with 3 μM of INDO-1AM dye (Molecular Probes; Eugene, Oreg.) incell media for 45 minutes at room temperature and washed with phosphatebuffered saline (PBS). After INDO-1AM loading, the cells are resuspendedin flux buffer (Hank's balanced salt solution (HBSS) and 1% FBS).Calcium mobilization is measured using a Photon Technology Internationalspectrophotometer (Photon Technology International; New Jersey) withexcitation at 350 nm and dual simultaneous recording of fluorescenceemission at 400 nm and 490 nm. Relative intracellular calcium levels areexpressed as the 400 nm/490 nm emission ratio. Experiments are performedat 37° C. with constant mixing in cuvettes each containing 10⁶ cells in2 mL of flux buffer. The chemokine ligands may be used over a range from1 to 100 nM. The emission ratio is plotted over time (typically 2-3minutes). Candidate ligand blocking compounds (up to 10 μM) are added at10 seconds, followed by chemokines at 60 seconds (i.e., C5a; R&DSystems; Minneapolis, Minn.) and control chemokine (i.e., SDF-1α; R&DSystems; Minneapolis, Minn.) at 150 seconds.

3. Chemotaxis Assays

Optionally, compounds may be further assayed for their ability toinhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μmpore polycarbonate, polyvinylpyrrolidone-coated filters in 96-wellchemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxisbuffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands(i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluatecompound mediated inhibition of C5aR mediated migration. Otherchemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used asspecificity controls. The lower chamber is loaded with 29 μl ofchemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the topchamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambersare incubated 1.5 hours at 37° C., and the number of cells in the lowerchamber quantified either by direct cell counts in five high poweredfields per well or by the CyQuant assay (Molecular Probes), afluorescent dye method that measures nucleic acid content andmicroscopic observation.

C. Identification of Inhibitors of C5aR

1. Assay

To evaluate small organic molecules that prevent the C5a receptor frombinding ligand, an assay was employed that detected ¹²⁵I-C5a binding tocells expressing C5aR on the cell surface (for example, cAMP stimulatedU937 cells or isolated human neutrophils). For compounds that inhibitedbinding, whether competitive or not, fewer radioactive counts areobserved when compared to uninhibited controls.

Equal numbers of cells were added to each well in the plate. The cellswere then incubated with radiolabeled C5a. Unbound ligand was removed bywashing the cells, and bound ligand was determined by quantifyingradioactive counts. Cells that were incubated without any organiccompound gave total counts; non-specific binding was determined byincubating the cells with unlabeled ligand and labeled ligand. Percentinhibition was determined by the equation:% inhibition=(1−[(sample cpm)−(nonspecific cpm)]/[(totalcpm)−(nonspecific cpm)])×100.

2. Dose Response Curves

To ascertain a candidate compound's affinity for C5aR as well as confirmits ability to inhibit ligand binding, inhibitory activity was titeredover a 1×10⁻¹⁰ to 1×10⁻⁴ M range of compound concentrations. In theassay, the amount of compound was varied; while cell number and ligandconcentration were held constant.

D. In Vivo Efficacy Models

The compounds of interest can be evaluated for potential efficacy intreating a C5a mediated conditions by determining the efficacy of thecompound in an animal model. In addition to the models described below,other suitable animal models for studying the compound of interest canbe found in Mizuno, M. et al., Expert Opin. Investig. Drugs (2005),14(7), 807-821, which is incorporated herein by reference in itsentirety.

1. Models of C5a Induced Leukopenia

a) C5a Induced Leukopenia in a Human C5aR Knock-in Mouse Model

To study the efficacy of compounds of the instant invention in an animalmodel, a recombinant mouse can be created using standard techniques,wherein the genetic sequence coding for the mouse C5aR is replaced withsequence coding for the human C5aR, to create a hC5aR-KI mouse. In thismouse, administration of hC5a leads to upregulation of adhesionmolecules on blood vessel walls which bind blood leukocytes,sequestering them from the blood stream. Animals are administered 20ug/kg of hC5a and 1 minute later leukocytes are quantified in peripheralblood by standard techniques. Pretreatment of mice with varying doses ofthe present compounds can almost completely block the hC5a inducedleukopenia.

b) C5a induced Leukopenia in a Cynomolgus Monkey Model

To study the efficacy of compounds of the instant invention in anon-human primate model model, C5a induced leucopenia is studied in acynomolgus model. In this model administration of hC5a leads toupregulation of adhesion molecules on blood vessel walls which bindblood leukocytes, hence sequestering them from the blood stream. Animalsare administered 10 ug/kg of hC5a and 1 minute later leukocytes arequantified in peripheral blood.

c) Mouse Model of ANCA Induced Vasculitis

On day 0, hC5aR-KI mice are intravenously injected with 50 mg/kgpurified antibody to myeloperoxidase (Xiao et al, J. Clin. Invest. 110:955-963 (2002)). Mice are further dosed with oral daily doses ofcompounds of the invention or vehicle for seven days, then mice aresacrificed and kidneys collected for histological examination. Analysisof kidney sections can show significantly reduced number and severity ofcrescentic and necrotic lesions in the glomeruli when compared tovehicle treated animals.

d) Mouse Model of Choroidal Neovascularization

To study the efficacy of compounds of the instant invention in treatmentof age related macular degeneration (AMD) the bruch membrane in the eyesof hC5aR-KI mice are ruptured by laser photocoagulation (Nozika et al,PNAS 103: 2328-2333 (2006). Mice are treated with vehicle or a dailyoral or appropriate intra-vitreal dose of a compound of the inventionfor one to two weeks. Repair of laser induced damage andneovascularization are assessed by histology and angiography.

2. Rheumatoid Arthritis Models

a) Rabbit Model of Destructive Joint Inflammation

To study the effects of candidate compounds on inhibiting theinflammatory response of rabbits to an intra-articular injection of thebacterial membrane component lipopolysaccharide (LPS), a rabbit model ofdestructive joint inflammation is used. This study design mimics thedestructive joint inflammation seen in arthritis. Intra-articularinjection of LPS causes an acute inflammatory response characterized bythe release of cytokines and chemokines, many of which have beenidentified in rheumatoid arthritic joints. Marked increases inleukocytes occur in synovial fluid and in synovium in response toelevation of these chemotactic mediators. Selective antagonists ofchemokine receptors have shown efficacy in this model (see Podolin, etal., J. Immunol. 169(11):6435-6444 (2002)).

A rabbit LPS study is conducted essentially as described in Podolin, etal. ibid., female New Zealand rabbits (approximately 2 kilograms) aretreated intra-articularly in one knee with LPS (10 ng) together witheither vehicle only (phosphate buffered saline with 1% DMSO) or withaddition of candidate compound (dose 1=50 μM or dose 2=100 μM) in atotal volume of 1.0 mL. Sixteen hours after the LPS injection, knees arelavaged and cells counts are performed. Beneficial effects of treatmentwere determined by histopathologic evaluation of synovial inflammation.Inflammation scores are used for the histopathologic evaluation:1—minimal, 2—mild, 3—moderate, 4—moderate-marked.

b) Evaluation of a Compound in a Rat Model of Collagen Induced Arthritis

A 17 day developing type II collagen arthritis study is conducted toevaluate the effects of a candidate compound on arthritis inducedclinical ankle swelling. Rat collagen arthritis is an experimental modelof polyarthritis that has been widely used for preclinical testing ofnumerous anti-arthritic agents (see Trentham, et al., J. Exp. Med.146(3):857-868 (1977), Bendele, et al., Toxicologic Pathol. 27:134-142(1999), Bendele, et al., Arthritis Rheum. 42:498-506 (1999)). Thehallmarks of this model are reliable onset and progression of robust,easily measurable polyarticular inflammation, marked cartilagedestruction in association with pannus formation and mild to moderatebone resorption and periosteal bone proliferation.

Female Lewis rats (approximately 0.2 kilograms) are anesthetized withisoflurane and injected with Freund's Incomplete Adjuvant containing 2mg/mL bovine type II collagen at the base of the tail and two sites onthe back on days 0 and 6 of this 17 day study. A candidate compound isdosed daily in a sub-cutaneous manner from day 0 till day 17 at aefficacious dose. Caliper measurements of the ankle joint diameter weretaken, and reducing joint swelling is taken as a measure of efficacy.

3. Rat Model of Sepsis

To study the effect of compounds of interest on inhibiting thegeneralized inflammatory response that is associated with a sepsis likedisease, the Cecal Ligation and Puncture (CLP) rat model of sepsis isused. A Rat CLP study is conducted essentially as described in FujimuraN, et al. (American Journal Respiratory Critical Care Medicine 2000;161: 440-446). Briefly described here, Wistar Albino Rats of both sexesweighing between 200-250 g are fasted for twelve hours prior toexperiments. Animals are kept on normal 12 hour light and dark cyclesand fed standard rat chow up until 12 hours prior to experiment. Thenanimals are split into four groups; (i) two sham operation groups and(ii) two CLP groups. Each of these two groups (i.e., (i) and (ii)) issplit into vehicle control group and test compound group. Sepsis isinduced by the CLP method. Under brief anesthesia a midline laparotomyis made using minimal dissection and the cecum is ligated just below theileocaecal valve with 3-0 silk, so the intestinal continuity ismaintained. The antimesinteric surface of the cecum is perforated withan 18 gauge needle at two locations 1 cm apart and the cecum is gentlysqueezed until fecal matter is extruded. The bowel is then returned tothe abdomen and the incision is closed. At the end of the operation, allrats are resuscitated with saline, 3 ml/100 g body weight, givensubcutaneously. Postoperatively, the rats are deprived of food, but havefree access to water for the next 16 hours until they are sacrificed.The sham operated groups are given a laparotomy and the cecum ismanipulated but not ligated or perforated. Beneficial effects oftreatment are measured by histopathological scoring of tissues andorgans as well as measurement of several key indicators of hepaticfunction, renal function, and lipid peroxidation. To test for hepaticfunction aspartate transaminase (AST) and alanine transaminase (ALT) aremeasured. Blood urea nitrogen and creatinine concentrations are studiedto assess renal function. Pro-inflammatory cytokines such as TNF-alphaand IL-1 beta are also assayed by ELISA for serum levels.

4. Mouse SLE Model of Experimental Lupus Nephritis.

To study the effect of compounds of interest on a Systemic LupusErythematosus (SLE), the MRL/lpr murine SLE model is used. TheMRL/Mp-Tmfrsf6^(lpr/lpr) strain (MRL/lpr) is a commonly used mouse modelof human SLE. To test compounds efficacy in this model male MRL/lpr miceare equally divided between control and C5aR antagonists groups at 13weeks of age. Then over the next 6 weeks compound or vehicle isadministered to the animals via osmotic pumps to maintain coverage andminimize stress effects on the animals. Serum and urine samples arecollected bi-weekly during the six weeks of disease onset andprogression. In a minority of these mice glomerulosclerosis developsleading to the death of the animal from renal failure. Followingmortality as an indicator of renal failure is one of the measuredcriteria and successful treatment will usually result in a delay in theonset of sudden death among the test groups. In addition, the presenceand magnitude of renal disease may also be monitored continuously withblood urea nitrogen (BUN) and albuminuria measurements. Tissues andorgans were also harvested at 19 weeks and subjected to histopathologyand immunohistochemistry and scored based on tissue damage and cellularinfiltration.

5. Rat Model of COPD

Smoke induced airway inflammation in rodent models may be used to assessefficacy of compounds in Chronic Obstructive Pulmonary Disease (COPD).Selective antagonists of chemokines have shown efficacy in this model(see, Stevenson, et al., Am. J. Physiol Lung Cell Mol. Physiol. 288L514-L522, (2005)). An acute rat model of COPD is conducted as describedby Stevenson et al. A compound of interest is administered eithersystemically via oral or IV dosing; or locally with nebulized compound.Male Sprague-Dawley rats (350-400 g) are placed in Perspex chambers andexposed to cigarette smoke drawn in via a pump (50 mL every 30 secondswith fresh air in between). Rats are exposed for a total period of 32minutes. Rats are sacrificed up to 7 days after initial exposure. Anybeneficial effects of treatment are assessed by a decrease inflammatorycell infiltrate, decreases in chemokine and cytokine levels.

In a chronic model, mice or rats are exposed to daily tobacco smokeexposures for up to 12 months. Compound is administered systemically viaonce daily oral dosing, or potentially locally via nebulized compound.In addition to the inflammation observed with the acute model (Stevensenet al.), animals may also exhibit other pathologies similar to that seenin human COPD such as emphysema (as indicated by increased mean linearintercept) as well as altered lung chemistry (see Martorana et al, Am.J. Respir. Crit. Care Med. 172(7): 848-53.

6. Mouse EAE Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a model of humanmultiple sclerosis. Variations of the model have been published, and arewell known in the field. In a typical protocol, C57BL/6 (Charles RiverLaboratories) mice are used for the EAE model. Mice are immunized with200 ug myelin oligodendrocyte glycoprotein (MOG) 35-55 (PeptideInternational) emulsified in Complete Freund's Adjuvant (CFA) containing4 mg/ml Mycobacterium tuberculosis (Sigma-Aldrich) s.c. on day 0. Inaddition, on day 0 and day 2 animals are given 200 ng of pertussis toxin(Calbiochem) i.v. Clinical scoring is based on a scale of 0-5: 0, nosigns of disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limbparalysis; 4, forelimb weakness or paralysis; 5, moribund. Dosing of thecompounds of interest to be assessed can be initiated on day 0(prophylactic) or day 7 (therapeutic, when histological evidence ofdisease is present but few animals are presenting clinical signs) anddosed once or more per day at concentrations appropriate for theiractivity and pharmacokinetic properties, e.g. 100 mg/kg s.c. Efficacy ofcompounds can be assessed by comparisons of severity (maximum meanclinical score in presence of compound compared to vehicle), or bymeasuring a decrease in the number of macrophages (F4/80 positive)isolated from spinal cords. Spinal cord mononuclear cells can beisolated via discontinuous Percoll-gradient. Cells can be stained usingrat anti-mouse F4/80-PE or rat IgG2b-PE (Caltag Laboratories) andquantitated by FACS analysis using 10 ul of Polybeads per sample(Polysciences).

7. Mouse Model of Kidney Transplantation

Transplantation models can be performed in mice, for instance a model ofallogenic kidney transplant from C57BL/6 to BALB/c mice is described inFaikah Gueler et al, JASN Express, Aug. 27, 2008. Briefly, mice areanesthetized and the left donor kidney attached to a cuff of the aortaand the renal vein with a small caval cuff, and the ureters removed enblock. After left nephrectomy of the recipient, the vascular cuffs areanastomosed to the recipient abdominal aorta and vena cava,respectively, below the level of the native renal vessels. The ureter isdirectly anastomosed into the bladder. Cold ischemia time is 60 min, andwarm ischemia time is 30 min. The right native kidney can be removed atthe time of allograft transplantation or at posttransplantation day 4for long-term survival studies. General physical condition of the miceis monitored for evidence of rejection. Compound treatment of animalscan be started before surgery or immediately after transplantation, e.g.by sub cut injection once daily. Mice are studied for renal function andsurvival. Serum creatinine levels are measured by an automated method(Beckman Analyzer, Krefeld, Germany).

8. Mouse Model of Ischemia/Reperfusion

A mouse model of ischemia/reperfusion injury can be performed asdescribed by Xiufen Zheng et al, Am. J. Pathol, Vol 173:4, October,2008. Briefly, CD1 mice aged 6-8 weeks are anesthetized and placed on aheating pad to maintain warmth during surgery. Following abdominalincisions, renal pedicles are bluntly dissected and a microvascularclamp placed on the left renal pedicle for 25-30 minutes. Followingischemia the clamps are removed along with the right kidney, incisionssutured, and the animals allowed to recover. Blood is collected forserum creatinine and BUN analysis as an indicator of kidney health.Alternatively animal survival is monitored over time. Compound can beadministered to animals before and/or after the surgery and the effectson serum creatinine, BUN or animal survival used as indicators ofcompound efficacy.

9. Mouse Model of Tumor Growth

C57BL/6 mice 6-16 weeks of age are injected subcutaneously with 1×105TC-1 cells (ATCC, VA) in the right or left rear flank. Beginning about 2weeks after cell injection, tumors are measured with calipers every 2-4d until the tumor size required the mice are killed. At the time ofsacrifice animals are subjected to a full necropsy and spleens andtumors removed. Excised tumors are measured and weighed. Compounds maybe administered before and/or after tumor injections, and a delay orinhibition of tumor growth used to assess compound efficacy.

What is claimed is:
 1. A compound having the formula:

or a pharmaceutically acceptable salt thereof; wherein each R¹ isindependently selected from the group consisting of halogen, —CN,—R^(c), —NR^(a)R^(b) and —OR^(a), and wherein each R^(a) and R^(b) isindependently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl, orwhen attached to the same nitrogen atom can be combined with thenitrogen atom to form a pyrrolidine ring; each R^(c) is independentlyselected from the group consisting of C₁₋₈ alkyl, C₁₋₈ haloalkyl andC₃₋₆ cycloalkyl, and wherein the aliphatic and cyclic portions of R^(a),R^(b) and R^(c) are optionally further substituted with from one tothree hydroxy, methyl, amino, alkylamino and dialkylamino groups; andoptionally when two R¹ substituents are on adjacent atoms, are combinedto form a fused five or six-membered carbocyclic ring; R² is a memberselected from the group consisting of H and F; p is 1; and R³ isX⁴R^(j), wherein X⁴ is C₁₋₃ alkylene, and R^(j) is pyrrolidinylsubstituted with CF₃.
 2. A compound of claim 1, wherein said compoundhas the formula:


3. A compound of claim 1, selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 4. A compound of claim 1,selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.